KR20010031416A - Cold-shock regulatory elements, constructs thereof, and methods of use - Google Patents

Abstract

Regulatory elements of the 5 'UTR of a cold shock induction gene are disclosed. This factor prolongs the expression of cold shock induction genes under conditions that elicit cold shock responses in bacteria, inhibits the expression of cold shock induction genes under physiological conditions, or cold shock induction conditions under conditions that elicit cold shock responses in bacteria. Improve their translation. Vectors and methods of protein expression with these elements are disclosed.

Description

Cold-shock regulatory elements, constructs thereof and methods of use

Inhibition of bacterial gene expression may include regulation of transcription or regulation of synthesis of mRNA from specific genes; Translational regulation, inhibition of the efficiency with which mRNA is translated into polypeptide sequences by ribosomes; And mRNA stability, or efficiency at which the number of specific mRNAs in the cell are degraded and inactivated. Controlling bacterial gene expression under conditions of physiological stress that elicit bacterial cold shock responses includes inhibition at all three levels described above.

Strict regulation of the expression of a small number of genes that function to function and function under stress conditions. For example, when bacterial cells are exposed to temperatures higher than their normal physiological temperature, a series of genes, called heat shock genes, are expressed. Such reactions to elevated temperatures are known and described in the prior art. Conversely, when bacterial cells are exposed to temperatures lower than their physiological temperature, a series of genes called cold shock (cs) genes are expressed. By expressing cold shock genes, cells first adapt to physiological stress and then grow under conditions of physiological stress. The present invention relates to a specific process for inhibiting the expression of cryogenic shock genes.

When the culture of E. coli is transferred from 37 ° C. to 15 or 10 ° C., many proteins, called cold shock proteins, are transiently induced during the growth lag period (Jones et al., 1987: Review, see, Thieringer et al., 1998). Yamanaka et al., 1998). CspA consisting of 70 amino acid residues was identified as a major cold shock protein (Goldstein et al., 1990) and its three-dimensional structure was analyzed by X-ray crystallography (Schindelin et al. 1994) and nuclear magnetic resonance analysis (Newkirk et al. 1994; Feng et al. 1998). Was determined to consist of five antiparallel beta-chain structures. cspA has been proposed to bind single-stranded DNA and RNA without high sequence specificity and to function as an RNA chaperone at low temperatures (Jiang et al. 1997).

Today, more than 50 CspA homologue proteins have been found in many protozoa. In addition, the sites of the so-called cold shock domains of the eukaryotic bio Y-box protein families such as human YB-1 and Xenopus FRgY-2 share 40% identity with E. coli cspA (for review, see Wolffe et al. 1992), which means that the low temperature shock zone is well preserved during the evolutionary process. In E. coli, nine genes cspA to cspI encoding cspA-type proteins have been found (see Yamanaka et al. 1998). Of these, cspA, cspB and cspG are cold shock fluids (Goldstein et al. 1990; Lee et al., 1994; Nakashima et al., 1996) and it is interesting that cspD is induced during stationary and malnutrition (Yamanaka and Inouye, 1997). . It has been reported that the E. coli broad-spread cspA group may have a function to respond to other environmental stresses (see Yamanaka et al., 1998).

cspA expression is transiently induced upon cold shock during a growth retardation period called the acclimation phase. It is believed that this period is required for the cells to adapt to new environmental conditions. Indeed, during the acclimation period CsdA (Jones et al., 1996), Proteins involved in the translation process, such as A (Jones and inouye, 1996) and CspA (Goldstein et al. 1990), are specifically produced, which play an important role in improving translation efficiency for non-low temperature shock proteins at low temperatures. (Review for Yamanaka et al. 1998). Among these cold shock proteins, cspA has been investigated very extensively for its mechanism of cold shock induction (for review, Yamanaka et al. 1998). The cspA promoter is very active at this temperature even though CspA is rarely detected at 37 ° C. (Fang et al. 1997; Mitta et al. 1997). Even though the cspA promoter is replaced with the lpp promoter, the major constitutive promoter, cspA expression is still cold shock inducible (Fang et al., 1997), which suggests that cspA induction at low temperatures is primarily at the mRNA stability and translational level. Shows that it happens. The cspA promoter, however, contains an AT-rich upper element (UP element) over the -35 site (Fang et al., 1997; Golenberg et al. 1997; Mitta et al. 1997), which is believed to play an important role in efficient transcription initiation at low temperatures. do. It has been found that cspA mRNA becomes very stable upon cold shock, indicating that mRNA stability plays a critical role in inducing cold shock of cspA (Branya et al. 1996; Goldenberg et al. 1996; Fang et al. 1997).

An important and unique feature of cspA mRNA is the unusually long 5'-untranslated site (5'-UTR) of 159 bases (Tanabe et al. 1992). This feature is also found in other Class I low temperature genes, such as cspB (Etchegaray et al. 1996) and cspG (Nakashima et al. 1996), which are dramatically induced after temperature reduction (see Thieringer et al., 1998 for review). This 5'-UTR is considered to play a decisive role in inducing cold shock of cspA (Brandi et al. 1996; Jiang et al. 1996; Goldenberg et al. 1996; Bae et al. 1997; Fang et al. 1997; Goldenberg et al. 1997; Mitta et al. 1997). .

Furthermore, a 14-base downstream box (DB), located 12 bases downstream of the translation initiation codon of cspA mRNA, partially complementary to a site called the anti-downstream box of the 16S rRNA (Sprengart et al. 1996). ) Has recently been found to play an important role in efficient translation at low temperatures (Mitta et al. 1997). This site of the RNA sequence designated as the downstream box is complementary to bases 1469-1483 in E. coli 16S rRNA (inverse-DB sequence). The formation of a double helix between the database and inverse i-DB is believed to be related to translation enhancement. The DB sequence also relates to the translation of λ cI mRNA, an mRNA that lacks untranslated sites and SD sequences (Shean and Gottesman 1992; Powers et al. 1988). Interestingly, λ cI translation is improved at 42 ° C. in temperature sensitive strains where the amount of ribosomal protein S2 is reduced at 42 ° C. In S2 deficient ribosomes, reverse-DB sequences become more accessible to the DB, resulting in improved translation initiation of λ cI mRNA. However, the DB role in the initiation of λ cI translation has been disputed by Resch and its partners (Resch et al. 1996). These authors tested the DB function by constructing a lacZ translational fusion with the λcI gene. The removal of six bases, including some DB sequences, did not reduce the formation of the translation initiation complex, and thus disputes the presence of the DB. In spite of this difficult role of DB (Sprengart and Porter 1997), we found that the presence of the DB sequence plays an important role in the translational effect in cold shock mRNAemfdp, before the induction of cold ribosomal factor (Mitta). It has been suggested to be involved in the formation of a stable starting complex at low temperatures. Thus, cspA expression is suppressed in a complex fashion at the level of transcription, mRNA stability and translation.

Here, a series of deletion variants were constructed at the 5'-UTR of cspA and their effects on cspA expression were analyzed by examining the amount of mRNA, mRNA stability and translational efficiency. In addition to mRNA stability, 5'-UTR has been found to play a major role in the translational efficiency of cspA mRNA, enhancing cspA expression upon cold shock. Specific sites of the 5'-UTR were found to mediate the inhibition process described above.

The present invention relates to the regulation of bacterial gene expression, and particularly to the inhibition of bacterial gene expression under conditions of physiological stress. More specifically, the present invention relates to inhibiting bacterial gene expression under conditions of physiological stress that induces cold shock responses of bacteria.

1 shows the effect of cspA upstream sites on plasmid pJJG78 and chromosomal cspA expression and synthesis of other cellular proteins: (A) pJJG78, (B) cspA upstream sites on chromosomal cspA expression and synthesis of other cellular proteins. Effect.

2 shows prolonged expression of CspA and inhibition of cold shock adaptation by pJJG78 and pUC19-600.

3 shows the deletion analysis of cspA upstream sites for cspA inhibitory function and inhibition of cold shock adaptation.

4 shows transcript levels from chromosomes and plasmid cspA.

5 shows the transcriptional conditions of the 5 'untranslated region of cspA mRNA for prolonged expression of cspA and inhibition of cold shock adaptation.

6 shows the effect on the production of other cold shock proteins and non cold shock proteins following overproduction of the 5 ′ untranslated region of cspA mRNA.

Figure 7 shows the effect on cold shock response according to the co-overproduction of cspA with the 5 'untranslated site of cspA mRNA.

8 shows the effect on CspA production following overproduction of the first 25 nucleotide sequences of cspA 5 ′ UTR.

9 shows cold induction of beta-galactosidase: (A) Construction of cspA-lacZ fusions. Wild type cspA is shown at the top. The cspA-lacZ fusion is shown from each 5 'end of the cspA promoter up to lacZ in each expression plasmid. The nucleotide number is +1 and starts at the transcription start position determined by Tababe et al. Cross hatched, open, spotted and deep bars indicate the cspA promoter, its 5 'untranslated site, cspA coding site, and lacZ coding site, respectively. The bold boxes indicate the SD sequences. The location of the missing site is shown by the nucleotide number. (B) Induction patterns of various deletion constructs. In the middle of the logarithmic growth phase, a culture of E. coli Ar137 containing various plasmids was transferred from 37 ° C. to 15 ° C. Samples were taken at 0.1, 2, 3, 5, 7 and 10 hours after transfer and beta-galactosidase activity was measured. cspA-lacZ fusion: pMM67 (o); pMM022 (●); pMM023 (□); pMM024 (■); pMM025 (Δ); And pMM026 (▲).

10 shows an analysis of mRNA stability. (A) Primer extension analysis of cells containing cspA-lacZ fusions. In the middle of the logarithmic phase, the culture of E. coli Ar137 containing various plasmids was transferred from 37 ° C to 15 ° C. To measure mRNA stability at 37 ° C., incubate at 15 ° C. for 30 minutes, transfer back to 37 ° C., and add Rifampicin to the culture until a final concentration of 200 ug / ml. RNAs were extracted after 0 (lane 1), 1 (lane 2), 3 (lane 3), and 5 minutes (lane 4) after rifampicin addition. To measure mRNA stability at 15 ° C., rifampicin was added 1 hour after cold transfer of rifampicin and RNAs were added 0 (lane 5), 5 (lane 6), 10 (lane 7), and 20 after addition of rifampicin. Extracted after minutes (lane 8). Primer extension was performed. (B) Graph of the results for 37 ° C. and 15 ° C. shown in (A). The radioactivity of the transcript was measured using Phosphoimager and was prepared with 0% at 0% using the transcript: pMM022 (●); pMM023 (□); pMM024 (■); pMM025 (Δ); And pMM026 (▲).

11 shows analysis of mRNA levels and translation efficiency. (A) Primer extension analysis of cspA-lacZ fusions. In the middle of the logarithmic phase, the culture of E. coli Ar137 containing various plasmids was transferred from 37 ° C to 15 ° C. RNAs were extracted from the culture after 0 hours (lane 1), 0.5 hours (lane 2), 1 hour (lane 30, 2 hours (lane 4), and 3 hours (lane 5) after the temperature drop shift at 37 ° C. Primer extension was performed as described (Mitta et al. 1997) (B) Graph of relative amounts of mRNA from the radioactivity of transcripts shown in (A) using pMM67 transcript at 37 ° C. as 1 The amounts were calculated: Relative amounts are shown at the top of each column: column 1, 0 hours; column 2, after 0.5 hours; column 3, after 1 hours; column 4, after 2 hours; column 5, after 3 hours. C) Relative translational efficiency of cspA-lacZ mRNA The translational efficiency at 15 ° C. was calculated by dividing the increase in beta galactosidase activity by the amount of mRNA using the following formula for the first two hours after cold shock: [(Gal 2h) x (OD 2h)-(Gal 0h) x (OD 0h)] / (avg. MRNA) where (Gal 0h) and (Gal 2h) are the temperature, respectively. Then beta-galactosidase activity after 0 and 2 hours and (OD 0h) and (OD 2h) are absorbances at 600 nm of the aliquots 0 and 2 hours after temperature transition, respectively; and (avg. MRNA) Is the average of the mRNA levels that were present 0.5, 1, and 2 hours after the transduction, and the relative translational efficiency of each mRNA was calculated with 100% of the mRNA efficiency of pMM 67. Column 1, pMM67; pMM022; column 3, pMM023; column 4, pMM024; column 5, pMM025; and column 6, pMM026.

12 shows the sequence similarity of cspA, cspB, cspG and cspI mRNAs of SD sequence attention and potential base binding between cspA mRNA and 16S rRNA. The nucleotide numbers of cspA (Tanabe et al. 1992), cspB (Etchegaray et al. 1996), cspG (Nakashima et al. 19960 and cspI mRNA (Wang et al. 1999) are given as +1 at the major transcription initiation sites. The same nucleotides in the three csp mRNAs are shown in bold: 13-base cognate sequences in cspA, cspB, cspG and cspI are boxed (upstream box) Location of SD sequence and initiation codon The potential base pair bonds of cspA mRNA and 16S rRNArks are indicated by vertical lines, and the locations of sites sensitive to RNase VI (powers et al., 1988) are indicated by dotted lines.

13 shows the role of the 13-base upstream box sequence at the cspA 5′-UTR site in cspA-lacZ expression. (A) Preparation of capA-lacZ fusions. Constructs were drawn in the same manner as shown in FIG. 9A except for a box of wavy lines representing the 13-base upstream box sequence 5'-GCCGAAAGGCACA-3 '(SEQ ID NO: 48) upstream of the SD sequence. pKNJ37 is identical to pMM007 except that the 13-base sequence is deleted. In both pMM007 and pKNJ37, cspA is translated fused to lacZ. pKNJ38 is identical to pKM67 (Mitta et al. 19970, except that a 13-base sequence was added by replacing the DNA fragment between XbaI and HindIII with synthetic oligonucleotides. In both pKM67 and pKNJ38, cspA is translated to lacZ translationally. (B) cold shock induction of beta-galactosidase cspA-lacZ fusions: pMM007 (o); pKNJ37 (o); pKM67 (o); and pKNJ38 (o).

FIG. 14 compares secondary structure of 5′-UTRs for deletion constructs. The secondary structure of the 5'-UTR of each deletion construct was predicted using a nucleotide sequencing program (DNASIS-Mac; Hitachi Software Enginnering Co. Ltd) based on the method of Zuker and Stieger, 1982. Nucleotides were numbered in cspA mRNA starting with transcription initiation site +1. Deletion positions in each variant are indicated by arrows using the nucleotide numbers of the deletion site. The highly conserved 13-base sequence of the SD sequence designated as the top box is boxed. Initiation codons and SD sequences are also boxed.

15 shows cspB translation enhancement by DB. (A) cspB-DB-inverse-DB complementarity: The cspB-DB sequence is boxed and covers the range of codons 5-9 (Mitta et al. 1997). Possible base pair binding between additional cspB mRNA-16S rRNA downstream of the DB is also shown. The AUG codons are circled, the SD sequences are boxed and the L-shaped arrows indicate the position at which the cspB gene was lacZdp fused (B) Translation cspB-lacZ fusion construct. At the top, the E. coli cspB gene is shown from its 5 'end. For pB3, pB13 and pB17, the lacZ gene is fused with cspB at residues +177 (3aa), +200 (13 aa) and +212 (17aa). pB 13sd and pB17sd are identical to pB13 and pB17 except that their SD sequences were converted from 5′-AGGA-3 ′ to 5′CTTC-3 ′, respectively. (C) Beta-galactosidase activity of cspB-lacZ constructs obtained before (time 0) and after (between 1, 2 and 3) temperature transition from 37 to 15 ° C. E. coli AR137 cells were transfected with pB3, pB13, pB13sd, pB17 and pB17sd and grown on them, then in the middle of logarithmic period (OD ₆₀₀ = 0.4), the cultures were transferred from 37 to 15 ° C and beta-galacto Sidase activity was measured. (D) mRNA levels of pB3, pB17 or pB13sd after temperature transition from 37 to 15 ° C .: cspB-lacZ mRNA was detected by primer extension before temperature reduction (time 0) and 1, 2 and 3 hours after temperature transition. (E) mRNA stability from pB3, pB13, pB17 and pB13sd: E. coli AR137 cells transformed with pB3, pB13, pB17sd were grown under the following conditions. In the middle of the logarithmic growth phase, the culture was transferred to 15 ° C. and after 30 minutes, rifampicin was added at a final concentration of 0.2 mg / ml (time 0). Total RNA was extracted 5, 10 and 20 minutes after rifampicin addition. cspB-lacZ mRnAs were detected by primer extension.

16 shows the effect of a fully bound DB to enhance the translation of cspA. (A) Translation cspA-lacZ fusion construct. The cspA gene structure is shown from the 5 'end up. pJJG78DB1 and pJJG78DB2 were constructed from pJJG78 as described in the experimental procedure. The DB sequences of pJJG78DB1 (12 pairs) and pJJG78DB2 (15 pairs) are shown below. (B) Beta-galactosidase activity of cspA-lacZ fusion constructs after cold shock at 15 ° C. E. coli AR137 cells transformed with pJJG78, pJJG78DB1 or PJJG78DB2 were grown in LB medium, and in the middle of the logarithmic growth phase (OD ₆₀₀ = 0.4), the cultures were transferred from 37 ° C to 15 ° C. Beta-galactosidase activity was measured before (transmission time 0) and 1, 2, and 3 hours post-transition. (C) deletion of cspA-lacZ mRNA. Total RNA was extracted and used as a template for primer extension as described above from E. coli AR 137 cells containing pJJG78, pJJG78DB1 or PJJG78DB2. (D) mRNA stability from cspA-lacZ constructs. E. coli AR137 cells transformed with pJJG78, pJJG78DB1 and pJJG78DB2 were grown as described above. In the middle of the logarithmic growth phase, the cultures were transferred to 15 ° C. and 30 minutes later rifampicin was added at a final concentration of 0.2 mg / ml (time 0). Total RNA was extracted 5, 10 and 40 minutes after rifampicin addition. cspA-lacZ mRNA was detected by primer extension.

17 shows that the complete pair DB improves translation at 37 ° C. (A) pIN-lacZ construct. XbaI-SalI fragments from pJJG78 or pJJG78DB2 were inserted into the XbaI-SalI site of pIN-III to make pINZ and pINZDB1 and used to prepare pINZDB2, pINDB3 and pINZDB4, respectively. (B) Beta-galactosidase activity of the pINZ-lacZ construct. E. coli AR 137 cell cultures transformed with pINZ, pINZDB1, pINZDB2, pINZDB3 and pINZDB4 were grown at 37 ° C. under the same conditions described in FIG. 1. IPTG (1 mM) was added to each culture in the middle of the logarithmic phase. Beta-galactosidase activity was measured before IPTG addition (time 0) and after 0.5, 1, 2 and 3 hours. (C) mRNA sequence of pIN-lacZ construct showing the location of SD, AUG and DB. lacZ at pJJG78 has a 10-pair DB. The complete DB pair located after the fifth codon has 16 residues complementary to the inverse DB. (D) shows the beta-galatosidase synthesis rate of the pINZ-lacZ preparation. Dilutions of E. coli AR137 cells containing pINZ or pINZDB1 were grown at 37 ° C. under the conditions described above. IPTG (1 mM) was added to each culture in the middle of the logarithmic growth phase. Beta-galactosidase synthesis rates were measured before IPTG addition (time 0) and after 0.5, 1, 2, 3 and 4 hours. The cells were pulsated-labeled with trans- [ ³⁵ S] -methionine. Cell extracts from each time point were analyzed by 5% SDS-PAGE and beta-galactosidase by phosphomes 6jd was measured. The beta-galactosidase synthesis ratios of pINZ and pINZDB1 are shown at each time point.

18 shows the ribosomal fraction of E. coli JM83 transformed with pINZ or pINZDB1. Ribosome particles were isolated as described by Dammel and Noller 1995. Cultures of E. coli JM83 containing pINZ or pINZDB1 were grown at 37 ° C. in LB medium containing 50 mg / ml ampicillin. In the middle of the logarithmic phase (OD ₆₀₀ = 0.4) 1 mM IPTG was added to each culture. Clomamphenicol (0.1 mg / ml) was added 15, 30 and 60 minutes after IPTG addition. Cell extracts were then spread on top of 5-40% (w / w) sucrose constructs. Polysomes and ribosomal units were separated by centrifugation at 15 ° C. for 2.5 hours at 4 ° C. Polysome Profiles were Detected Using the FPLC System 0.2 ml from each fraction (0.5 ml) was transferred onto nitrocellulose membranes using the Minifolod II Slot-Blot System (Schleicher and Schuell). lacZ mRNA was detected by hybridization using [ ³² P] -labeled M13-47. Phosphoriser values from the hybridization are shown on the right. pINZ and pINZDB1 mRNAs are indicated by blocked and open squares, respectively.

19 Enhancement of translation by fully binding DB at 37 ° C. (A) Measurement of pINZ and pINZDB1 mRNAs. Cultures of E. coli JM83 containing pINZ or pINZDB1 were incubated at 37 ° C. under the conditions described in FIG. Total RNA extracted 15, 30 and 60 minutes after IPTG (1 mM) cjar was used as a template for primer extension according to the procedure described above. (B) Beta-galactosidase activity of pINZ and pINZDB1 in a multi-copy expression system. E. coli JM83 transformed with pINZ or pINZDB1 were grown at 37 ° C. under the conditions described in FIG. 20A. Beta-galaltosidase activity was measured before (hour 0) and 0.5, 1, 1.5, 2 and 2.5 hours (open circles and squares) before addition. Blocked circles and squares indicate activity in the absence of IPTG.

20 shows cell free0 synthesis of beta-galactosidase from pINZ and pINZDB1. (A) pINZ or pINZDB1 DNA (160 ng; 1 ul) was extracted from E. coli 30S extract (20 ul) (Promega) And a transcription-translational binding reaction, lane 1, pINZ DNA; lane 2, pINZDB1 DNA; and lane 3, control reaction without added DNA.Samples were precipitated with acetone and analyzed by 15% SDS-PAGE. Production of beta-galactosidase was detected (B) Time course in vitro synthesis from pIN and pINZDB1 was performed as described above Samples were taken after incubation at 37 ° C. for 15, 30, 60 and 120 minutes. (C) Each reaction from each of the time course experiments described above was run in duplicate using non-radioactive methionine, transferred to nitrocellulose membrane and hybridized with [ ³² P] labeled M13-47 oligonucleotide.

22 shows enhanced translation of pINZDB1 in cells using S2-deficient ribosomes. (A) Beta-galactosidase activity from pINZ and pINZDB1. E. coli CS240 and CS239 (Shean and Gottesman 1992) were transformed with pINZ or pINZDB1 and grown at 30 ° C. in LB medium. In the middle of the logarithmic phase the cells were transferred to 42 ° C. in the presence of 1 mM IPTG. Beta-galactosidase activity was measured in Miller units prior to transition to 42 ° C. (hours) and after 0.5, 1, 1.5, 2.5 and 3.5 hours. (B) Induction of relative lacZ expression in cells of pINZDB1 and pINZrks using S2-deficient ribosomes. Before the transition to 42 ° C. (time 0), the rate of beta-galactosidase expression from pINZ and pINZDB1 at CS239 for CS240 was determined as 1 and thus the rate after transition to 42 ° C. was calculated.

Summary of the Invention

The present invention encompasses isolated nucleic acid molecules that prolong the expression of cold shock inducing genes under conditions of physiological stress leading to cold shock in bacteria.

The invention also encompasses isolated nucleic acid molecules that inhibit the expression of cold shock inducing genes under physiological conditions.

The invention also encompasses isolated nucleic acid molecules that enhance the translation of cold shock inducing genes under conditions of physiological stress that elicit cold shock responses in bacteria.

Three essential factors that regulate the suppression of genes expressed under conditions of physiological stress that elicit a cold shock response in bacteria, mediated by the 5'-untranslated region (5'-UTR) of mRNA transcripts encoding cold shock inducing proteins The process was found. Special regions of the 5'-UTR of cold shock mRNA transcripts (A) transient nature of cs gene expression after cell exposure to physiological stresses that induce a cold shock response, (B) inhibition of cs gene expression at physiological temperature, And (C) mediate translation enhancement of cold shock induced mRNA transcripts by ribosomes.

DNA constructs encoding such inhibitory elements alone or in combinations thereof or in combination with other inhibitory sequences known in the art, such as promoters, downstream boxes, and transcription termination sequences, may be used to induce physiological induction of bacterial low temperature shock responses. It can be used for prolonging the expression of cs gene or heterologous gene under conditions, improving the translation efficiency and inhibiting the expression of these genes under normal physiological conditions. This may result in hyper-expression of the desired protein product, which may be unstable at physiological temperature, improperly overlapped or present in an inclusion body in the host cell at physiological temperature, or may be disrupted by the host cell. expression)

The present invention also provides the advantage that the translation of normal cell proteins is inhibited under conditions in which the preferred protein product is overexpressed. This allows the desired product to accumulate in the host cell and simplify the separation of the desired product from the host cell polypeptide.

Accordingly, the present invention also encompasses transgenic bacteria having a vector comprising one or more of the aforementioned elements and a transgenic bacterium having one or more of the aforementioned elements and a vector having the gene sequence of interest for expression. Such target sequences may be cold shock gene sequences or heterologous genes.

As reported by Sprengart et al., The bacterial downstream box (DB) plays an important role in the translation of mRNA producing proteins. The DB binds to a portion of the membrane 16S rRNA near the 3 'end and places the mRNA and rRNA in appropriate relative positions for translation to occur.

During the time when ADB binds to the DB of overexpressed mRNA, the 16S rRNA cannot participate in the translation of cellular mRNA rather than the bound overexpressed mRNA. Bacterial whole protein production machinery can be suspended by supplying the bacteria with an mRNA that coats the DB complementary to the ADB of the 16s RNA that binds all or substantially all of the 16S rRNAs.

The term ″ complementary ″ as used herein includes ″ substantially complementary ″. Thus, the term ″ complementary ″ does not require complete complementarity. The two sequences are `` complementary '' as defined by Kahl, Dictionary of Gene Technology VCH Publisher, InC (1995), incorporated by reference. That is, it is complementary if the two nucleotide sequences can form a double helix that is hydrogen bonded to each other according to the Watson-Crick base binding law. Two complementary RNA sequences or RNA and DNA sequences form a bond of A-U, G-C or G-U. ″ Complete complementarity ″ is not needed.

As used herein, "homolog" refers to a molecule that has a molecular sequence that is substantially the same as the referenced nucleic acid sequence, but may include an additive, a deletion or a substituent. Homologous molecules are defined as such molecules that hybridize with reference nucleic acid molecularly precisely complementary nucleic acids under low or high stringency conditions and perform the same function as the referenced nucleic acid.

For example, and not by way of limitation, low stringency conditions for hybridization are: a filter containing DNA is run at 40 ° C. for 6 hours at 35% formaldehyde 5 × SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, Preheat in a solution containing 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20 × 10 ⁶ cpm ³² P-labeled probe do. The filter is then incubated in the hybrid mixture at 40 ° C. for 18-20 hours and washed in a solution containing 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS at 55 ° C. for 1.5 hours. do. This wash solution is replaced with a fresh wash solution and incubated another 1.5 hours at 60 ° C. The filter is blotted dry and exposed to perform radiography. Preferably or if necessary, a third washing step is carried out at 65-68 ° C. for 1.5 hours and the filter is reexposed to the film.

For example, and not by way of limitation, high stringency conditions are as follows: the filter containing the nucleic acid to be probed was run at 65 ° C. for 8 hours to overnight, 6 × SSC, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.02. Prehybridization is performed in a buffer containing% PVP, 0.02% Ficoll, 0.02% BSA and 500 μg / ml denatured salmon sperm DNA. The filter is hybridized in a prehybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5-20 × 10 ^{6 32} P-labeled probes at 65 ° C. for 48 hours. Washing of the filter was carried out in a solution containing 2 × SSC, 0.01 PVP, 0.01% Ficoll and 0.01% BSA for 1 hour at 37 ° C., followed by washing in 0.1 × SSC for 45 minutes at 50 ° C. The filter is blotted dry and exposed to perform autoradiography. Alternative methods for high and low stringency hybridization, well known in the art, may be used alternatively.

As noted, the ADB is a nucleotide sequence of about 14 bases located at the 3 'end portion of the 16S rRNA, near the decoding site of the 16S rRNA. Known bacteria 16S rRNA nucleotide sequences are known and can be found in the GenBank database. Thus, for a selected bacterium, the ADB can be readily identified by comparing the sequence of the ADB to a known bacterium, eg, E. coli. Once the ADB is identified, a DB complementary to the ADB can be prepared and allowed to be contained in the appropriate mRNA as described below.

The mRNA of the present invention is mRNA isolated or mRNA transcribed from the isolated DNA. The mRNA comprises a codon that is an initiation codon, preferably AUG. Other suitable start codons for the mRN include GUG and UUG.

MRNAs of the invention also typically comprise a downstream box sequence that is 3 'to the initiation codon. The codon of the DB may or may not be in-frame with the initiation codon. The DB sequence can be right next to the initiation codon so that there are no intervening nucleotides. In general, the DB is spaced from the initiation codon by interfering nucleotides between 1 and 30 nucleotides in size. The base sequence of the interference sequence is not critical and may consist of any nucleotide sequence. Preferably, the interfering nucleotide sequence is 7 to 15 nucleotides in size and most preferably 12 nucleotides in size. In addition, the DB may overlap with the start codon. That is, any one of the three nucleotides of the start codon of the mRNA of the present invention may form the 5 'end of the DB.

The DB sequence of the mRNA of the present invention is a nucleotide sequence complementary to the ADB of the bacterial 16S rRNA. Although the DB may be at least 20 bases, in general, the DB is 6 to 20 bases in size and preferably 8 to 14 bases in size. For example, the DB may contain nucleotides that are complementary to nucleotides that are 3 ', 5' or both in ADB. Regardless of the length of the DB, higher complementarity between the DB and ADB is associated with more effective annealing, which results in more effective inhibition of bacterial protein synthesis, according to the method of the present invention.

In addition to the start codon, the DB and any interfering sequences, the mRNA constructs of the invention may comprise a nucleotide sequence at 5 'of the start codon or at 3' of the DB.

For example, the mRNA construct may comprise a sequence at 3 'of the DB encoding the polypeptide or include a stop codon. Likewise, the mRNA construct may contain an untranslated sequence and / or a shine-dalgano sequence at initiation codon 5 '.

Except for any additional nucleotides at the 5 'or 3' end, the length of the mRNA construct, including the starting todon, any interfering sequence, and the DB, is between 8 nucleotides and 45 nucleotides in length. Of course, if the mRNA comprises a 5 'or 3' sequence in addition to the necessary components, such as the Shine-Dalgarno sequence, the mRNA can be longer to hundreds of nucleotides in length.

Preferably, although not required, the mRNA construct lacks an enzyme cleavage site for RNA endonucleases. Particularly preferably there is no enzymatic cleavage site for the RNA endonuclease in the portion of the mRNA construct comprising the essential portion of the construct, ie the start codon and the DB, otherwise the mRNA construct will be degraded and the bacterial 16S rRNA Freely binds to bacterial mRNA.

MRNA constructs of the invention may have sequences that are similar or identical to mRNA sequences found naturally in bacteria. For example, mRNAs for several cold cooling proteins, such as mRNAs for the E. coli protein CspA, CspB, CspG, CsdA and RbfA, are substantially complementary to the ADB of the shine-dalgano sequence, the start codon and the E. coli 16S rRNA. Includes a downstream box. Other E. coli mRNAs, including the Shine-Dalgarno sequence, initiation codons and downstream boxes complementary to E. coli ADB, include RecA, Hns, NusA, InfB and CspD.

Non-limiting examples of suitable DBs for mRNA constructs are shown below. Each DB below is substantially complementary to the ADB of E. coli 16S rRNA with ADB.

ADB 3 '(-1481) UACUUAGUGUUUCA (-1469) 5' (SEQ ID NO: 1)

DB # 1: 5'AUGACUGGUAUCGU3 '(SEQ ID NO: 2)

DB # 2: 5'AUGACUGGUUUCGU3 '(SEQ ID NO: 3)

DB # 3: 5'AUGACUGGUUUAGU3 '(SEQ ID NO: 4)

DB # 4: 5'AUGAGUUAUGUAGA3 '(SEQ ID NO: 5)

DB # 5: 5'AUGGCGAAAAGAAU3 '(SEQ ID NO: 6)

Suitable mRNA preparations according to the present invention can be prepared using any of the above or other suitable DBs. For example:

5'AUGX _(n) AUGACUGGUAUCGU3 '(SEQ ID NO: 7)

Wherein n is the total number from 0 to 30, X is G, C, U or A and each X occurrence is the same or not the same as another X occurrence. Alternatively, the 5 'end of the DB overlaps the start codon.

The DNA of the present invention is isolated DNA encoding mRNA suitable for the mRNA preparation of the present invention. The DNA may further comprise additional nucleotide sequences comprising a promoter sequence towards the 5 'direction of the start codon. The promoter sequence can be used to regulate transcription of mRNA preparations. The DNA may comprise a sequence having a function other than a promoter such as a shine-dalgano sequence in the 5 'direction of the initiation codon and / or a sequence having an unknown action. The DNA may comprise a stop codon sequence in the 3 'direction of the portion encoding the DB of the mRNA preparation, or a sequence encoding a polypeptide and a sequence required for transcription termination.

An example of a DNA suitable for encoding an mRNA preparation of the invention is 5'ATGY (n) ATGACTGGTATCGT3 '(SEQ ID NO: 8), where n is the total number from 0 to 30, Y is G, C, T or A, and each Y occurrence is the same or not the same as another Y occurrence. Alternatively, the 5 'end of the DB overlaps the start codon ATG. DNA may include additional sequences at the 5 'and / or 3' ends of the DNA.

DNA sequences of the invention can be included in mediators or cloning vectors, such as plasmids or phage vectors. The DNA sequence in the vector can be located under the control of a promoter sequence located in the 5 'direction of the start codon. These vectors, including the DNA of the present invention, can be used to transform host bacteria that can be used to overexpress the mRNA of the present invention, ie bacteria transformed to higher levels than those produced in similar untransformed bacteria. MRNA is prepared. Any bacterium that can be transformed by the cloning vector is suitable as a host of the DNA sequences of the present invention. Methods of making cloning vectors and transforming bacteria are well known in the art and are described, for example, in Ausubel et al. ″ Recent Molecular Biology Protocols ″, J. Wiley & Sons, Inc. (1995).

Overexpression of the mRNA sequences of the present invention results in the production of mRNA at levels higher than those normally expressed in bacteria. Depending on the extent of mRNA overexpression, the production of bacterial proteins is inhibited. If mRNA is expressed at high levels, bacterial protein production will be completely stopped, ultimately causing the bacteria to die.

Therefore, preparations that produce mRNA are useful as antibiotics that inhibit or kill bacterial growth. mRNA production preparations can be filled into bacteriophage, which allows the mRNA to be used as an infection prevention or topical antibiotic. The delivery strategy is thought to be designed to allow transformation of bacteria that cause food or animal infections, such as humans, dogs, cats, cattle, horses and poultry.

According to the method of the present invention, mRNAs comprising the initiation codon and the DB complementary to the ADB of the bacterial 16S rRNA are induced to be overexpressed in the bacterium and then annealed with the ADB of the bacterial 16S rRNA, by another mRNA in the bacteria. Suppresses the production of encoded proteins.

Any means of delivery that cause overexpression of the mRNA of the invention is suitable for the method of the invention. For example, the bacteria can be transformed by a medium containing a DNA sequence encoding the mRNA of the present invention.

If desired, expression of the mRNA sequences of the invention is controlled by placing the DNA sequence under the control of an inducible promoter. For example, if it is necessary to kill harmful bacteria or inhibit growth while competing for beneficial bacteria, the DNA sequence can be placed under promoter control that is only reactive to products present only in the first bacteria.

Another means of controlling the expression of protein production-inhibiting mRNA sequences is to use DNA sequences encoding mRNAs that are unstable under certain conditions.

For example, the mRNA 5 ′ untranslated region (5′UTR) of E. coli cold shock protein CspA comprises a 5 ′ region of the shine-dalgano region that is sensitive to degradation by RNase E at a temperature of about 37 ° C. Therefore, cspA mRNA containing 5'UTR has a half-life of about 12 seconds and is unstable under normal growth conditions. Other cold shock proteins such as E. coli CspB and CsdA are unstable at menstrual growth temperatures due to their mRNA instability. Under low temperature shocks, such as cooling to 15 ° C., the half-life of cspA mRNA increases rapidly up to about 15 minutes, which is 75 times the mRNA stability at normal physiological growth temperature.

This region, or 5'UTR of cspB or csdA mRNA, can be used to modulate the expression of the mRNA sequence of the present invention, as mRNA containing the 5'UTR of cspA mRNA is indeterminate at 37 ° C. Antibiotic effect is only exerted at or below physiological growth temperature. Cold shocked bacteria require new ribosomal elements, and their synthesis is inhibited by overproduction of mRNA containing DN sequences, so the antibiotic effect of the method of the present invention is enhanced under cold shock conditions.

The antibiotic effect of the method of the present invention, wherein the mRNA of the present invention is caused to be overexpressed in bacteria, increases with an increase in the number of copies of the mRNA to be expressed. That is, minimal overexpression of the mRNA of the present invention inhibits protein production by bacteria, while the inhibition will not be sufficient to further inhibit the growth of bacteria or kill the bacteria. High levels of mRNA expression results are closely associated with increased protein production inhibition. If the copy number is high enough in bacteria, protein production will be completely suppressed.

Similar effects are known to be associated with ADB of bacterial 16S rRNA and DB complementarity of overexpressed mRNA. Overexpression of mRNA containing DB with 100% complementarity will bind more efficiently with ADB compared to mRNA containing DB with lower (75%) complementarity. Therefore, the protein inhibitory effect of mRNA with higher DB complementarity will be more pronounced compared to mRNA with lower DB complementarity. Therefore, when using mRNA with lower DB complementarity, obtaining the same or similar antibiotic effect as compared to having mRNA with higher DB complementarity would be useful for expressing mRNA with higher copy number.

In addition, the translation inhibition properties of the downstream box are advantageous for overexpression of heterologous genes in transformed bacteria after cold shock. Inhibition of translation of endogenous bacterial proteins causes high levels of heterologous gene products to accumulate in the transformed organism. In addition, preparations comprising a strong promoter and a downstream box coupled with the 5 'untranslated site of the cold shock inducing gene, which act to stabilize mRNA transcription under reduced temperature, will efficiently regulate the expression of high levels of heterologous genes under inspiration. .

Another important embodiment of the present invention relates to the role of the 5'-terminal untranslated region of mRNA for cspA, the major cold shock protein of E. coli in cold shock adaptation.

When the incubation temperature of Escherichia coli cells growing in log phase is changed from 37 ° C. to 10 ° C., there is a lag period before starting cell growth again (Jones et al., 1987). Similar to the high temperature shock reaction, E. coli responds to temperature drops by inducing a specific type of gene expression called the low temperature shock reaction, including a series of protein induction called the low temperature shock proteins (Jones et al. 1992; Jones and Inoue 1994). . The cold shock response occurs during the period of adaptation of cell growth, which is considered to be required for cellular adaptation to low temperatures.

CspA, the major cold shock protein in Escherichia coli, is induced rapidly when temperature drops, and its synthesis reaches 13% of total protein synthesis (Goldstein, 1990). However, CspA production during the cold shock reaction is temporary and drops to low levels when cell growth begins at low temperatures. CspA consists of 70 amino acids and is 43% identical to the cold shock domains of eukaryotic Y box proteins known to be involved in gene regulation and mRNA masking (Wolfe et al., 1992; Wolfe, 1993). The tertiary structure of CspA was measured, consisting of five anti-parallel β plates forming β-pipe structures (New Kirk et al., 1994; Schindelin et al., 1994). The two RNA binding sites RNP1 and RNP2 are identical to the β2 and β3 plates. Seven of the eight fragrant residues are located on the same surface, and single-stranded DNA is linked to these surface aromatic residues (Newkerk et al., 1994). There is also a suggestion that CspA acts as an RNA chevron to increase translation efficiency at low temperatures (Jones and Inoue, 1994).

Cold box

In one embodiment of the invention, the DNA sequence can sustain the transient expression of cold shock genes while bacteria adapt to the physiological stresses that cause cold shock reactions. Therefore, the temporal nature of the cold shock reaction and normal transient expression of the cold shock inducing gene are suppressed and the expression continues at a high level for about 2 to 3 hours, for a longer time. DNA sequences capable of conferring such activity include a 5'-UTR or at least a 5'UTR portion and promoter of a 5'-UTR that is active under physiological stress conditions that induce a cold shock response. In addition, it was found that the entire 5'-UTR is not needed, and that a sequence that inhibits the transient expression of a cold shock gene such as cspA is present in the first 25 nucleotides of the 5'-UTR.

Comparison of nucleotide sequences with these activities with other cold shock genes revealed that cspB, cspG and csdA have similar sequences in their 5'-UTR and expressed in a transient manner in response to physiological stresses that induce a cold shock response. Indicates that This means that the genes are regulated in a similar way to cspA. Based on this sequence comparison, it can be seen that the sequences involved in the activity, hereinafter referred to as cold boxes, are located between +1 and +11 nucleotides of cspA, cspB, cspG and csdA 5'-UTR.

Coldboxes normally function to down-regulate the expression of cold shock genes. This was revealed by hyperexpression of cspA 5-UTR in Escherichia coli which leads to prolongation of E. coli cold shock gene expression. Coldboxes probably interact with the CspA protein itself or with another protein whose action depends on CspA. This was confirmed through observation of inhibition of hyperexpression and continuous expression of CspA protein in E. coli. Therefore, the cold box is likely to contain a repressor binding site that acts to suppress the expression of the cold shock gene after induction of the cold shock gene, thereby causing the transient expression of the gene. As CspA protein levels increase after gene induction, CspA initiates interactions with coldbox by directly binding to coldbox sequences or by indirectly stimulating another factor that interacts with coldbox and inhibits cold shock induction genes. cause.

Low temperature shock gene expression refresher at physiological temperature

5'UTR is unique because it is longer than most E. coli UTRs. The 5'UTR is responsible for several functions in cspA expression. It inhibits cspA expression at 37 ° C. This different activity was mapped through the analysis and preparation of defined deletions through the 5'-UTR site of Yamanaka et al. The sequence involved in cspA expression suppression at 37 ° C is between +56 and +117 of cspA 5'-UTR (see Yamanaka et al., Unpublished manuscript). For optimal inhibition it seems likely to require other sequences in the 5'-UTR. In other words, the effect of removing the +56 to +117 sites is important and does not take into account the overall level of inhibition observed with complete 5'UTR. Therefore, he also concluded that other sequences also play an important role in inhibiting cspA expression at 37 ° C. However, the localization of the minimal inhibitory active moiety to a particular region of the 5'-UTR is novel. 5'-UTR begins to cleave into the action domain and the +56 to +117 sites represent new sites of action.

UTRs also have a positive effect on mRNA stability at 15 ° C. This was measured through the increase to similar stability levels of mRNA preparations with UTRs. However, although a large number of mRNAs were present in the experiment, mRNAs are not translated unless a DB is present.

Therefore, the effect of increasing mRNA stability and increasing mRNA translation is mediated by sequences associated with cspA transcription. However, certain sequences that mediate RNA stability have not been well characterized at this site.

Improved translation of cold shock mRNA

5'-UTR enhances the translation of cspA transcription through sequences located between +117 and +143. Comparison of these sites for different cold shock induction revealed that 13 base sequences (+ 123- + 135) with the 5'-GCCGAAAGGCACA-3 'sequence were present in both cspA, cspB and cspGdp and improved translation. Therefore, the sequence mediates efficient mRNA translation. These 13 base sites show similarity to 16S rRNA similar to the previously observed DB, and the new translational enhancement site is complementary to different sites of 16S rRNA rather than DB. Therefore, this 5'-UTR site also aids csp mRNA translation with a downstream box-like mechanism that interacts with ribosomal RNA.

In another embodiment of the invention, expression plasmids have been prepared that allow expression at high levels of cryoshock inducing or heterologous genes. The expression plasmid comprises a DNA fragment encoding a 5′UTR or cold shock inducing gene region comprising one or more regulatory elements located downstream of a promoter that operate under physiological stress conditions inducing cold shock responses in bacteria. The promoter may be selected from the group comprising the cspA, cspB, cspG or csdA promoter, E. coli lpp promoter, or other promoters active under physiological stress conditions. The expression plasmid may also include DNA fragments encoding the 5′-UTR and transcription termination sequences, including one or more regulatory elements downstream of the promoter. Examples of transcription termination sequences are well known in the art and include sequences such as the popular strain rrnB terminator.

Expression plasmids of the invention may also comprise a restriction site or multiple restriction sites, said sites, or sites located between 5'-UTR encoding one or more regulatory elements and transcription terminators to facilitate insertion of heterologous genes. have.

Finally, expression plasmids of the invention may comprise additional sequences known in the art to facilitate efficient transcription of the expression gene. The sequence may comprise a DNA fragment encoding a shine-dalgano sequence located between the 5′UTR sequence and a restriction site, a shinedallano sequence and / or a downstream box located between the restriction sites. The source of the shinedalgano sequence is not particularly limited and may be derived from cold shock proteins or may be derived from other genes. The expression plasmid may be involved in the expression of high levels of heterologous genes for prolonged periods of time under physiological stress conditions causing a cold shock response of bacteria. Under these conditions, endogenous protein synthesis by the host bacteria is inhibited, which allows the heterologous gene product to accumulate at high levels in the cell.

In a preferred embodiment of the present invention, the expression vector comprises a promoter, 5′UTR, cold box, Shindalgano sequence and downstream box (DB). The expression vector can be used to allow the nucleic acid molecule encoding the protein of interest to be inserted into the expression vector using at least one restriction site.

In another embodiment of the invention, nucleic acid molecules encoding proteins that are unstable or improperly formed in bacterial host cells at physiological temperatures can be expressed at temperatures lower than the physiological temperature of the host cells. The nucleic acid molecule may preferably be inserted into a restriction site frame or at a site that allows nucleic acid transcription and mRNA translation. These conditions facilitate proper protein formation, increase stability or reduce the rate of degradation of the expression product.

Plasmids or vectors can be used to transform bacteria by any method known in the art, including, but not limited to, calcium chloride transformation, electropermeation, and the like. The type of bacteria to be transformed is also not particularly limited. As a preferred embodiment E. coli is used. Transgenic bacteria can be used to overexpress the desired protein or can be used to produce large quantities of plasmids or vectors for substantial isolation or purification.

In a preferred embodiment for the overexpression of the protein of interest, the nucleic acid molecule encoding the protein of interest uses one or more restriction sites, preferably if the start codon is upstream of the insertion site for the protein of interest, using an initiation codon frame. Inserted into plasmid. Alternatively, if the nucleic acid molecule encoding the protein of interest has its own initiation codon, the nucleic acid encoding the protein of interest can be inserted into the plasmid and the self initiation codon will be provided as an initiation codon for transcription.

An article of manufacture can be designed and made to include selective markers. That is, for example, there is a drug resistance gene that allows the transgenic bacteria to grow in the presence of a drug that inhibits the growth of the non-transgenic bacteria. Any optional marker known in the art can be used. Examples of selective markers include, but are not limited to, ampicillin resistance, neomycin resistance, kanamycin resistance, and tetracycline resistance.

The preparation may also include an induction promoter. Induction promoters can induce the synthesis of the protein of interest. For example, it may include, but is not limited to, a lacZ promoter that can be induced by adding IPTG to the culture.

In a preferred embodiment, the preparation will comprise a promoter having activity under conditions that induce a low temperature shock response in bacteria such as changing the temperature of the culture medium containing the transforming bacteria to 10-15 ° C. Preferably, overexpression of the target protein under conditions that induce a cold shock reaction results in reduced synthesis of one or more native bacterial proteins. More preferably, a large number of native protein synthesis is reduced or inhibited.

The following examples are provided to illustrate one aspect of the invention and should not be construed as limiting the scope of the invention as defined in the claims.

Example

Example 1: Plasmid Preparation

PJJG02 was prepared from pJJG01 (Goldenstein et al., 1990) by the following method: A 998-bp fragment containing the whole cspA gene was obtained from HindIII and XmnI cleavage from pJJG01. This fragment was then treated with a cleno fragment of DNA polymerase and inserted into the SmaI position of pUC9.

pJJG21 was prepared from pJJG02 by creating an XbaI site upstream of the following Shine-Dalgano sequence:

+13 AATTT (A) C (T) TAG (A) AGGTAA + 153 (SEQ ID NO: 9) (the native nucleotides in parentheses were replaced with underlined nucleotides). pJJG81 was prepared from pJJG02 by creating an XbaI site upstream of the transcription initiation site of cspA having the following sequence: + 1ACGGTTCTAGACGTA + 15 (SEQ ID NO: 10) (underlined nucleotide refers to insertion base)

pJJG78 is a transcription hybrid of the lacZ and 0.6 kb cspA upstream sites hybridized by the following method: The 1-kb EcoRI / BamHI fragment containing cspA from pJJG21 was filled with a Klenow enzyme and inserted into the SmaI site of pUC19. . The 0.6-kb XbaI fragment, including the cspA regulatory site (-457 to +143), is then cut and inserted into the XbaI site in pKM005 (Inoue, M et al., 1983) in the forward direction.

pUC 19-600 was constructed by inserting a 0.6-kb EcoRI / XbaI fragment into the EcoRI / XbaI site of pUC19 in pJJG21. PJJG81 / X, S comprising fragment 1 (Figure 3), was constructed by removing the 0.74-kb XbaI / SalI fragment from pJJG81. Both ends are treated with glenow fragments and then self-linked. All other structures shown in FIG. 3 are prepared by PCR (Beibermanheim Protocol). PCR extended fragments are inserted into the SmaI site of pUC19. All PCR products are identified by DNA sequence (Rang et al., 1977).

p2JTEK is constructed as follows. primer

And primer

The PCR product by is cloned into the SmaI site of pUC19. This PCR product contains a CSPA from -146 to +25 because the cspA transcription start site is defined as +1. Subsequently primers cspA transcription terminator

and

Expand by PCR using. PCR products are then digested using EcoRI and then cloned into the plasmids described above digested with EcoRI and SspI. 52-bp Kpn and EcoRI from BlueScript IISK are cloned into EcoRI and KpnI sites. All PCR products are identified by DNA sequence (Sang et al., 1977).

p6m TEK is the first PCR different primers; primers

And

It is written in the same way as p2JTEK except that The PCR product contains cspA because the cspA transcription start site is defined as +1. All PCR products are identified by DNA sequence (Rank et al., 1977).

Pulse-label experiments are performed by the method described above (Jiang et al., 1993). Proteins are analyzed either by polyacrylamide SDS-gel electrophoresis (Inoue et al., 1982) or by two-dimensional electrophoresis (John et al., 1987) as described above.

Each 5′-UTR deletion is introduced by two step PCR. As a first step, two PCRs are performed for each variant. One reaction is primer,

(nucleotides -67 to -49 of cpsA) and a variant primer # 4311 comprising a preferred variant sequence and assisting in cspA,

(Auxiliary +186 to +198) and also using a variant primer F comprising the same variant and in the same direction in cspA. The lower case is the outer nucleotide for constructing the NheI or BamHI site (underlined), and the value is given using the major transcriptional introductory at +1 and determined by Tanabe et al. (1992). Regarding the construction of pMM022, pMM023, pMM024, pMM025 and pMM026, primer DIR,

Primer D2R

Primer D3R,

Primer D4R,

And primer D5R,

Variant primer R and primer D1F,

And as the variant primer F, respectively, and the position of each deletion is indicated by a slash. A plasmid containing wild type cspA, pJJG02 (Goldstein et al., 1990) is used as template DNA. Each set of first PCR products is then mixed, denatured, annealed and expanded with Taq polymerase. Further expand the product using primer 67F and primer # 4311. The final PCR product is digested using NheI or BamHI and inserted into the XbaI-BamHI site of pKM005 (Inoue 1983). For pKNJ37, PCR fragments are cloned into pRS424X, pRS414 derivatives (Simons et al., 1987) that change the unique SmaI site to the Xbal site.

Regarding the construction of pMM007, it is carried out using primer 67F and primer # 4311 as primers and pJJG02 as template. The PCR fragment is digested with NheI or BamHI and inserted into the XbaI-BamHI site of pRS414X.

pKNJ38 was constructed as follows: Oligonucleotide # 8509,

Is first annealed and then pKM67 (Mita et al., 1997) is digested using Xbal and HindIII.

DNA sequences of all constructs are identified using DNA chain termination methods (Sang et al., 1977).

E. coli AR137 Harboring Different plasmids are grown at 37 ° C. and mid-log faces into M9-Casamino acid 15 using a 125 ml flask. The culture is transferred to a 15 ° C. shaking water bath. The culture temperature is raised from 15 ° C. to 37 ° C. for 2-3 minutes under the conditions used. 1.5 ml cultures are taken immediately before the temperature is downshifted (0 hours) and taken at 1, 2, 3, 5, 7 and 10 hours after the temperature is downshifted. Beta-galactosidase activity of the culture is measured according to the method of Miller (1972) et al. The analysis is performed in duplicate at each time point.

The cspB DNA fragment is expanded by PCR using synthetic oligonucleotide primers containing a BamHI site at the 5 'end. PCR fragments B3, B13 and B17 are prepared using the plasmid carrying the wild type cspB gene, pSJ7 (Lee et al., 1994) as template DNA. The 5'-terminal oligonucleotide primers used in each step of the PCR reaction are

The 3'-terminal oligonucleotide primers for PCR products B3, B13 and B17 are respectively

Deletion of the SD sequence from the cspB gene is prepared as site-directed mutagenesis using the QuickChange Trademark Site Directed Mutagenesis Kit (Stratagen). PCR reaction uses pSJ7 plasmid as template and oligonucleotide

To prepare pSJ7sd. pSJ7sd is used as a template to prepare PCR products B13sd and B17sd. The 5 'and 3' terminal oligonucleotide primers used in these PCR reactions are the same as those used in PCR fragments B13 and B17. All of the above PCR products were cloned at the BamHI site of the pRS414 vector (Simmons et al., 1987; Lee et al. 1944) to prepare pB3, pB13 and pB13sd, pB17 and pB17SD constructs.

cspA-lacZ fusion constructs can be prepared by inserting annealed oligonucleotides into the EcoRI site of pJJG78 (Jiang et al., 1996). Annealed Oligonucleotide DB1 And DB1 ' Or DB2 And DB2 ' PJJG78DB1 or pJJG78DB2 constructs can be prepared, respectively.

The pIN-lacZ fusion construct can be prepared by inserting XbaI-SalI fragments from pJJG78 or pJJG78DB2 into the XbaI-SalI site of pINIII to form pINZ and pINZDB1, respectively (Jiang et al., 1996). Then, the annealed oligonucleotide ZDB2

ZDB2 ' PINZDB2 can be prepared by inserting the XbaI-EcoRI site of pINZ.

Annealed Oligonucleotide ZDB3 And ZDB3 ' Or ZDB4 And DB4 ' PINZDB3 and pINZDB4 can be prepared by inserting into the XbaI-EcoRI site of pINZ, respectively.

β-galactosidase activity

E. coli AR137 (pcnB ⁻ ) (Harlocker et al., 1993) or JM83 (pcnB ⁻ ) harvesting different plasmids was treated at 37 ° C. in a 20 ml LB medium containing 50 μg / ml ampicillin in a 125 ml flask. Growth was to the mid-log stage. After rm, this culture was transferred to a 15 ° C. vibratory bath or isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. At each time point, 100 ml of culture was taken. Β-galactosidase activity was measured according to the method of Miller (Miller, 1972).

Example 2: Isolation of RNA, Extension of Primers and Ribosome Analysis

E. coli AR137, harvesting different plasmids, was grown under the same conditions used for the β-galactosidase assay described above. To determine the amount of cspA-lacZ mRNA, 1.5 ml cultures were taken at each time point and RNA was extracted by hot phenol extraction according to Sarmientos et al. (1983).

For the stability test of mRNA at 15 ° C., 1 hour after the temperature was lowered, rifampicin was added so that the final concentration was 200 μg / ml, and transcription was stopped, and 1.5 ml of the culture was taken at each time point. For the stability test of mRNA at 37 ° C, mRNA was accumulated by first treating at 15 ° C for 30 minutes. Next, 5 ml of culture was taken and mixed with 5 ml of medium containing 400 μg / ml rifampicin in a glass flask in a 37 ° C. vibratory bath. The medium was preheated to 60 ° C. Using this method the temperature of the culture was immediately raised from 32 ° C. to 37 ° C. within 1 minute.

³² P-labeled primer M13-47 by the primer extension method described above (Jiang et al.) CspA-lacZ mRNA was detected. The sequence is complementary to the 5'-terminal code sequence of lacZ. The product was separated on a modified polyacrylamide gel (6%) and quantified using Phosphorimager (BioRad).

Pulse labeling

Cultures of E. coli AR137 (pcnB ⁻ ) with pINZ or pINZDB1 were grown in 600 ml LB medium in a 4 liter flask under the same conditions as used for the β-galactosidase assay described above. In the mid-log step, IPTG (1 mM) was added to each culture. The ribosomal particles were isolated by Dammel and Noller's method (1995), but the method was modified as follows: 100 ml aliquots from the original culture were taken at each time point and chloramphenicol was added to give a final concentration of 0.1 mg /. The growth of the cells was stopped by bringing to ml. Cells were immediately collected by centrifugation (at 5000 × g for 10 min at 4 ° C.) and in buffer [20 mM Tris-HCl (pH 7.6), 10 mM MgCl ₂ , 6 mM β-mercaptoethanol and 1 mg / ml lysozyme]. Resuspend and then freeze for several hours at -80 ° C. Cells were hemolyzed by freeze-thaw (Ron et al., 1996). Cell extracts (0.5 ml) are then placed on top of a 5-40% (W / W) sucrose gradient (7.5 ml), and the polysomes and ribosomes subunits at 4 ° C. using a Becjman SW-41 rotor. Separation was performed by centrifugation at 151,000 × g for 2.5 hours. The polysomes were detected by FPLC system and a total of 15 fractions were collected, 0.5 ml each.

detection of lacZ-mRNA

From each polysome fraction (0.5 ml), Manifold 0.2 ml were spotted onto nitrocellulose membrane using II Slot-Blot System (Schleicher and Schuell). ^The lacZ-mRNA was detected by hybridization using ³² P-labeled M13-47 primers (S. Inouye and M. Inouye, 1991) and the amount was measured using Phosphorimager.

Translation under laboratory conditions

Using the E. coli S30 Extract System for Linear Templates Kit (Promega), the transcription-translation reaction was performed according to the manufacturer's protocol as follows: In 20 μl Pre-mix containing all amino acids except methionine, 10 μCi Trans- [ ³⁵ S] methionine (1,175 Ci / mmol; NEN Life Science Products) and E. coli S30 extract, 160 ng of pINZ or pINZDB1 (1 μl) were added and the mixture was incubated at 37 ° C. for 45 minutes. The product was precipitated and annealed by 15% SDS-PAGE.

Example 3: Multicopy Effect of cspA Upstream Region on Cold Shock

The cspA gene was induced immediately after the temperature had dropped from 37 ° C. to 15 or 10 ° C., and the CspA production rate was found to peak after the temperature had dropped to 15 ° C. after 1 hour and to 10 ° C. after 2 hours. (Goldstein et al., 1990). After this point, CspA production drops back to baseline levels. This temporary production period of CspA corresponds to the growth period known as the Jag period, which is observed after cold shock (Jones et al., 1987). Thus, it is believed that this transient expression of CspA is required for the adaptation of cells to low temperatures.

To characterize this transient expression of CspA, the present inventors sought to identify the regions required for the regulation of capA expression during the adaptation period. For this purpose, we first constructed pJJG78, where the 600 bp cspA upstream region is the lacZ gene. In a transcriptional manner (see FIG. 1). The cspA upstream region of 600 bp includes a region from -457 to +143, which is immediately before the shine-dalgano sequence of cspA because the cspA transcription initiation site is +1 (Goldstein et al., 1990). E. coli strain CL83 is transformed with pJJG78 and preparation of β-galactosidase was pulse-labeled with [ ³⁵ S] -methionine at 0, 0.5 and 3 hours after the temperature had dropped from 37 ° C. to 15 ° C. Examination by cells. As a control, CL83 cells alone as well as CL83 cells transformed with the vector pKM005 (Inouye, 1983) were used. It can be seen from FIG. 1B that the expression of cspA was highly induced for both CL83 and CL83 / pKM005 0.5 hours after the temperature was lowered (FIG. 1B, lanes 2 and 5). However, as described above (Goldstein et al., 1990), this high level of expression is transient and falls back to basal level at 3 hours (FIG. 1B, lanes 3 and 6). No cspA expression was detected at time point 0 (FIG. 1B, lanes 1 and 4), and no β-galactosidase was produced at all time points in both states (FIG. 1B, lanes 1-6).

In contrast to CL83 and CL83 / pKM005, the development of β-galactosidase was apparent in cells with pJJG78 (Fig. 1B, lanes 7-9), indicating that the 600bp upstream region of cspA was responsible for inducing cold shock. Imply sufficient. Surprisingly, the preparation of cspA is not transient and remains high at 3 hours after cold shock induction in cells with pJJG78 (Figure 1B, lane 9 compared to lanes 3 and 6). Since pJJG78 does not have a cspA code sequence, the high production rate of CspA 3 hours after cold shock induction is due to the chromosomal cspA gene. Under the conditions used, the chromosomal cspA gene does not appear to be inhibited, that is, inhibition is released. Interestingly, there is another band in the X-marked portion of Figure 1B, whose expression pattern is almost the same as that of cspA. It is a cold shock protein, the preparation of which is also inhibited by the presence of pJJG78. This cold shock protein X has recently been identified as CsdA associated with ribosomes (Jones et al., 1994).

Synthesis of most cellular proteins is blocked on a larger scale at low temperatures than in CL83 and CL83 / pKM005 for cells with pJJG78 (compare FIG. 1B, lanes 8 and 9 with lanes 2, 3, 5, 6). This result suggests that the adaptation of cells to low temperatures is impaired by a more severe cold shock response when the cells carry a multicopy plasmid with part of the csp gene.

Since the sustained synthesis of cspA after cold shock is induced by pJJG78, the 600 bp cspA upstream region cloned into pJJG78 may insulate the factor responsible for the inhibition of cspA production after cold shock, leading to the inferred that sustained expression or termination of cspA may result. can do. To test this hypothesis, the 600bp upstream region of cspA was cloned back into pUC19. The plasmid is referred to as pUC19-600. The number of copies of pUC19 (300 copies / cell) is 10 times greater than pJJG78 (30 copies / cell) derived from pBR322. Pulse labeling tests were performed as described above (Jiang et al., 1993). As shown in FIG. 2, in CL83 cells, CspA production was elevated after 1.5 hours at 15 ° C. and lowered to baseline levels after 3 hours (FIG. 2, lanes 1 to 6). CL83 cells with pJJG78 also observed CspA production after 24 hours at 15 ° C. (FIG. 2, lanes 7-12). The cspA production pattern in CL83 cells with pUC19-600 is similar to that for pJJG78 (FIG. 2, lanes 13 to 18). However, the expression level of cspA is much higher with pUC19-600 than with pJJG78, as judged from CspA preparation after 3-5 hours. Therefore, the greater the number of copies of cspA, the stronger the expression of cspA. In other words, CsdA (marked X) exactly shows the same expression pattern as CspA when looking at all lanes in FIG. 2.

As shown in Figure 1B, pJJG-containing cells showed specific inhibition of normal protein synthesis at low temperatures. (Comparing lenses 8 to 11 with lenses 2 to 5 in Figure 2, respectively.) Significantly, in terms of protein synthesis rate and inhibition time, this inhibition was more apparent in cells containing pUC19-600 than in cells containing pJJG78. . (Comparing lenses 14 to 17 in Figure 2 with lenses 8 to 11 in Figure 2.) The higher copy number of the cspA 600-bp upstream has a stronger inhibitory effect on other cell protein synthesis. This means that low temperature shock adaptation is suppressed.

Example 4: Transient Generation of 5 ′ Undenatured Untranslated Portions of cspA mRNA

A series of internal fragments were generated by PCR and cloned into the SmaI site of pUC19, as shown in Figure 3, to find the exact portion within 600-bp that is essential for cspA inhibition and cold shock adaptive inhibition at low temperatures. This arrangement is identified as a DNA sequence. The ability to inhibit CspA expression and to inhibit cold shock adaptation of each construct at 15 degrees was studied by pulsatile classification experiments. First, deletion mutations were generated at the 5 ′ end of the 600-bp fragment. As shown in Figure 3, fragment 3 (186-base clear), fragment 2 (312-base clear), fragment 2E (366-base clear), and fragment 2G (390-Q base clear) Kept the jersey function. Next, fragment 2 was split into fragments 2A and 2B, which matched by 23bp, as shown in Figure 3. Surprisingly, both 2A and 2B lost this feature. Fragment 2F, which is 33bp longer at the 5 'end than fragment 2B, was also generated, which remained functional. It has been found that cspA resistant constructs also result in inhibition of cold shock adaptation and vice versa.

The fact that fragment 2 has the function of cspA inhibition and cold shock adaptation, while fragment 2A does not suggest that the cspA promoter portion alone is not sufficient for the function of the 600 bp fragment. The fact that functional fragment 2G is 31bp longer at the 5 'end than nonfunctional fragment 2F suggests the possibility that both functions require a complete cspA promoter to simulate the 5'UTR of cspA mRNA. Note that cspA mRNA has an unmodified sequence of 159-bases at the 5 ′ end (Golden et al., 1990). To confirm this possibility, we studied cspA simulations generated from fragments cloned with primer extension (Fragments 2, 2A, 2B, 2E, 2F). Primer 3550 with primer extensions ranging from +124 to +143 in two independent primers-5`UTR using total RNA sections isolated from cells containing various plasmids incubated at 15 degrees for 1 hour. And a primer 3551- corresponding to the cspA coding sequence +224 to +243. The former primer found cspA mRNA simulated in plasmids and chromosomes, while the latter only found mRNA in the chromosomal cspA gene because the plasmid did not contain the cspA coding portion.

As shown in Figure 4, the amount of simulation from the chromosome cspA gene presented by Primo 3551 is basically the same in all constructs (Figure 4, lines 1-6). In contrast, the 5`UTR that Primer 3550 presents The amount of cspA simulations shows two different levels. In nonfunctional constructs (pUC19-2A, pUC19-2B, pUC-2F), the amount of simulation (3, 4, and 6 lines in Figure 4) found by Primer 3500 was almost identical to that of pUC19 (1 line in Figure 4). The same amount. This means that the part of cspA cloned into this plasmid has not been simulated. On the other hand, functional constructs (pUC19-2 and pUC19-2E) showed higher cspA simulations found by primer 3550 compared to pUC19 (line 1 in Figure 4) (lines 2 and 5 in Figure 4, respectively). These results demonstrate that 5`UTR of cspA mRNA is simulated in fragments 2 and 2E but not in fragments 2A, 2B and 2F. Therefore, the ability to prolong cspA expression and inhibit cold shock adaptation at low temperatures is closely correlated with 5′UTR simulation of cspA mRNA.

Total promoter fragments (-457 to -1) and 6 bases (+1 to +6) were cloned into pUC19 to clearly demonstrate that 5'UTR simulation of cspA mRNA is essential for cspA inhibition and cold shock adaptation inhibition. It became. This fragment refers to fragment 1 (see Figure 3). Thus, the 5'UTR of most cspA mRNAs was removed from fragment 1. The beat classification experiment shown in Figure 5B prevents fragment 1 from cspA despite the fact that the simulation from the cspA promoter was clearly found by the primer extension (Figure 5A). It is concluded that the +1 to +143 portions of 5`UTR should be simulated to influence cspA appearance and cold shock adaptation.

Example 5: Cold shock genes affected by 5`UTR transient production of cspA mRNA

Next, the transient generation of 5`UTR of cspA mRNA was studied to determine if cspA mRNA affected the appearance of other cold shock genes. The protein appearance of cold shock cells overproducing cspA 5′UTR was analyzed by two-sided electrophoresis. The plasmid pJJG21 / X, S has a 5`UTR of the entire cspA promoter and most cspA mRNAs (+1 to +143), and pJJG81 / X, S has a 5`UTR of the whole cspA promoter and cspA mRNA Hold only the first 6 base parts. The cells harboring these plasmids are labeled at the moment as previously described (Jian et al., 1993). At 37 degrees, the protein synthesis rate and protein form are very similar for both (Figure 6, A and B); note that no cold shock protein is found. When these cells are moved to a temperature of 15 degrees for one hour (Figure 6, C and D), the synthesis of cold shock proteins (1) cspA (2) CspB` (3) CspB (4) CsdA becomes very active. Note the prediction that CspB 'is accompanied by CspB and is either a modified form of CspB or an unidentified cold shock protein (Ezegarei et al., 1996). It is comparable. Although the synthesis of most other cellular proteins was significantly reduced in both than at 37 degrees, a stronger inhibitory effect was observed in cells modified with pJJG21 / X, S. When the cells were incubated for 3 hours at 15 degrees, most of the cellular protein synthesis returned to normal levels with the decrease of the total cold shock protein in the cells with pJJG81 / X, S (Figure 6F). On the other hand, for cells with pJJG21 / X, S, the production of total cold shock proteins (labeled 1 to 4) remained at very high levels and the production of other cellular proteins decreased (Figure 6E). 5'UTR overproduction of mRNA results in the inhibition of cspA as well as other cold shock genes, suggesting that the genes of cold shock proteins are regulated by general mechanisms. It was also confirmed that the suppression of cold shock adaptation was due to the 5′UTR overproduction of cspA mRNA that inhibited the synthesis of other cellular proteins.

Based on the results stated above, 5′UTR overproduction of cspA mRNA involves the inhibition of other cellular proteins. This means that the growth of cells upon cold shock has a more severe inhibitory effect on cells overproducing 5′UTR of cspA mRNA than in normal form cells. Growth of cells containing either pUC19-600 or pUC19-2G (see Figure 3) was indeed severely inhibited. This is caused by a long delay period. (no data)

Example 6: effects of cspA overproduction

The overappearance of 5'UTR of cspA mRNA results in prolonged overproduction of CspA (see Figure 2). Thus, the observed effect may be due to CspA overproduction rather than 5'UTR of cspA mRNA. This possibility can be experimented with CL83 cells carrying the entire cspA gene and containing pJJG02. The beat classification experiment proceeds as described above. As shown in Figure 7, cspA and csdA (protein X gene) with CL83 containing pUC19 were induced 1 hour after temperature change at 15 degrees (Lines 1, 2) and returned to baseline levels 3 hours after temperature change. (Line 3). On the other hand, when cells were transformed with pJJG02, the appearance of cspA was not only induced at 15 degrees, but also higher than cells containing pUC19 as measured by two-sided cleatin electrophoresis (as suggested). Note that this observation is observed at 3 hours at 15 degrees (line 6). There is no long delay period of cell growth despite the fact that 3 hours of CspA overproduction after cold shock is very similar to the case of cspA 5′UTR overproduction already presented. It is important to note that no delayed production of and other cold shock proteins such as CspB and CsdA was observed at the same time. This implies that the combined production of 5'UTR and CspA of cspA mRNA inhibited the effect of overproduction of 5'UTR only, and that high levels of CspA production at 3 hours after cold short were not the reason for this effect.

Example 7: Discovery of inhibitor bondage points

We sought to find the specific part responsible for cspA inhibition in the 5`UTR of cspA mRNA. Due to the 11 base sequences commonly found in 5'UTRs in cspA, cspB, and csdA, this portion including an array of positions from +1 to +25 of the 5'UTR of cspA mRNA was tested. This portion was placed under the control of the cspA promoter in pUC19 for p2JTEK construction. Cells containing p2JTEK were studied to examine the morphology of total protein synthesis 3 hours after a temperature change from 37 to 15 degrees. As shown in Fig. 3, the production of CspA and CsdA was clearly inhibited, and the inhibition of other cellular proteins was accompanied by a brighter background compared to the cells containing pUC19 (Fig. 8, line 1). Cells containing only simulated parts of cspA mRNA (Fig. 8, line 2) +1 to +6 and showed the same shape as pUC19 cells (Fig. 8, line 1). This indicates that part of the cspA inhibition is present in the first 25 base arrays of 5'UTR of cspA mRNA.

Example 8: Deletion analysis of cspA 5`UTR

We have already described cspA-lacZ fusion in which two lacZ genes are fused to cspA at +26 (pKM67; Mitta et al., 1997) or +143 (pJJG78; Jiang et al., 1996) of cspA mRNA. Configured. Beta-galactosidase activity of cells containing pJJG78 was very low at 37 degrees and increased about 10-fold at 2 hours after temperature change to 15 degrees. On the other hand, beta galactoidase activity of pKM67-containing cells was very high at 37 degrees (Mita et al., 1997). Possible function: (I) cspA expression inhibition at 37 degrees (II) mRNA stabilization at cold shock (III) denaturation efficiency at 15 degrees (Mitta et al., 1997)

To investigate which part of the 5'-UTR is responsible for the positive or negative regulation of cspA expression, we attempted a cleavage assay of 5'-UTR and examined the effects of various cleavage on cold-shock induction of cspA. Tested. For this purpose a series of 5′-UTR cleavage mutants were constructed, from which 26- to 32-base cleavages were made every about 30, and the resulting 5′-UTRs were transfused and fused at the 13th amino acid residue of cspA. lacZ. The resulting plasmids pMM022, pMM023, pMM024, pMM025 and pMM026 are +27 to +27, +28 to +55, +56 to +86, +86 to +117, +118 to +143 at 5'-UTR (Fig. 9A). Each includes a truncation mutation. The cleavage of +56 to +85 for pMM024 was originally designed, but analysis of the transformants included an external base cleavage at the +86 position. The wild type cspA-lacZ transition fusion construct plasmid pMM67 (Mita et al., 1997) was used as a control. E.coli AR137, pcnB mutants, known to retain pBR322 derivatives at low copy number (Rofilato et al., 1986), are used as a host of transformation to avoid the multiplexing effect of the genes constructed on the expression of pBR322 derivatives. .

Transformed cells are grown at 37 ° C. in M9-casamino acid medium and beta-galactosidase activity is measured after the temperature is reduced from 37 ° C. to 15 ° C. Beta-galactosidase activity at 37 ° C. (zero time point in FIG. 9B) was higher in cells harboring pMM024 (Δ56-86) and pMM025 (Δ86-117) than in cells harboring wild type pMM67. Other cleavage mutants [pMM022 (Δ2-27), pMM023 (Δ28-55) and pMM026 (Δ118-143)) show very low beta-galactosidase activity while they are 10 times higher. These results suggest that the 5'-UTR region of bases +56 to +117 is involved in the inhibition of cspA at 37 ° C. Interestingly, beta-galactosidase activity increased almost five-fold with pMM024 (Δ56-86) after temperature reduction, while only less than two-fold with pMM025 (Δ86-117), which was pMM025 (Δ86). The cleaved region at -117) suggests an important role in the cold-shock induction of cspA. Similar to pMM025 (Δ86-117), beta-galactosidase activity using pMM023 (Δ28-55) is hardly induced at low temperatures. In particular, since the beta-galactosidase activity is very low at both 37 ° C. and 15 ° C. (FIG. 9B), the region cleaved at pMM026 (Δ118-143) appears to play a decisive role both at high and low temperatures in cspA expression. Cleavage of the region of bases +2 to +27 (pMM022), including the cold-box sequence included in cspA autoregulation (Jiang et al., 1996), is expected to have little effect on cold-shock induction of cspA. none.

Example 9: Analysis of cspA-lacZ mRNA

As described above, all deletion mutations in the 5'-UTR except pMM022 (Δ2-27) affect the cold shock induction of cspA. The cspA promoter is known to be active even at 37 ° C. (Goldenberg et al., 1997; Fang et al, 1997; Mita et al., 1997). Since all deletion preparations have a complete cspA promoter (FIG. 9A), the transcriptional efficiency of these preparations will be the same. CspA mRNA stability, on the other hand, varies considerably depending on growth temperature (Brandy et al., 1996; Goldenberg et al., 1996; Breed, 1997; Fang et al., 1997; Goldenberg et al., 1997; Mita et al., 1997). Therefore, the effect of deletion mutations on cspA expression at low temperatures may be due to the different mRNA stability of the preparation. To investigate this portion, primer extension analysis was performed by quantifying cspA-lacZ amounts for each mutation at different times after addition of rifampicin at 37 ° C. and 15 ° C. (FIG. 10A). Strain AR137, a pcnB mutant, was used to avoid positive or negative multiplication effects. The amount of transcript at each reaction time was measured using a phosphoimator, and the remaining mRNA amount (percentage amount at zero time) was plotted as shown in FIG. 10B. All transcripts were unstable with a half life of 30 to 45 seconds at 37 ° C. However, at 15 ° C. they become very stable with half lives of 20 to 40 minutes. These half-lives are similar to those of the wild-type preparation pMM67 obtained earlier as well as the half-life of the wild type chromosome cspA (Pang et al, 1997; Goldenberg et al. 1997) (Mita et al., 1997). The β-galactosidase activity induced at 15 ° C. compared to the similar mRNA half-life at low temperatures varied for all preparations as shown in FIG. 9B.

The results indicate that cspA induction efficiency measured by β-galactosidase activity is not related to mRNA stability of each deletion preparation, but rather to mRNA amount and / or translation efficiency of each preparation. Therefore, the inventors of the present invention investigated the amount of mRNA for each preparation at different bases after cold shock using the primer extension method. The results are shown in FIG. 11A and the amount of transcript was measured with a phosphorizer. Their relative amounts were calculated with the amount of pMM67 transcript at zero time as 1 (FIG. 11B). At 37 ° C., the transcript amounts for pMM022 (Δ2-27) and pMM024 (Δ56-86) were similar to wild type preparation pMM67 (column 1 of FIG. 11B). It should be noted that the β-galactosidase activity of pMM024 (Δ56-86) at 37 ° C. was at least 10 times higher than pMM022 (Δ2-27) (FIG. 9B). For pMM023 (Δ28-55), pMM025 (Δ86-117) and pMM026 (Δ118-143), the amount of transcript at 37 ° C. was about half of pMM67. Again, it should be noted that the β-galactosidase activity of pMM025 (Δ86-117) was more than 10 times higher than pMM023 (Δ28-55) and pMM026 (Δ118-143) (FIG. 9B). This result indicates that there is no association between transcript amount and β-galactosidase activity at 37 ° C.

Example 10 Transcription Control by 5′-UTR

After shifting down the temperature, the amount and induction pattern of cspA-lacZ mRNA dramatically increased in the overall configuration is shown in FIGS. 11A and 11B. This indicates that the accumulation of transcript is very similar to that of wild type pMM67, as maximum induction is observed at 1 h after shifting the temperature down. The pattern of mRNA levels is very similar between pMM67 and pMM024 (Δ56-86), while the amount of mRNA is approximately half of pMM67 mRNA at each time point, but the other shows a similar induction pattern. Since the promoter activity of all deletion constructs is identically configured and further mRNA stability is very similar to that of wild type construct (FIG. 10B), the amount of mRNA for all deletion constructs except pMM024 (Δ56-86) may be low. Preferred is slower because the transcriptional extension ratio and / or transcriptional attenuation in the 5'-UTR is slow. It was surprisingly found that the amount of mRNA for pMM026 (Δ118-143) accumulated after cold shock was the same as for pMM022 (Δ2-27), pMM023 (Δ28-55) and pMM025 (Δ86-117) ( See FIG. 11B). Nevertheless, cold-shock induction of β-galactosidase activity is very low for pMM023 (Δ118-143) during cold-shock treatment, meaning that the mRNA for this construct is incompletely modified. . Relative strain efficiency at 15 ° C. is calculated for the overall configuration as an increase in β-galactosidase activity and amount of mRNA during cold shock (see brief description of Figures Legend in FIG. 11). As shown in FIG. 11C, the transformation efficiency of pMM026 (Δ118-143) mRNA is very poor and is calculated to be 9.5% for pMM67 mRNA (FIG. 11C 6 column). Thus, it is clear that deletion of +143 at base +118 of the 5'-UTR negatively affects the deformation. Except for pMM026 (Δ118-143), the modification efficiencies of mRNAs of pMM023 (Δ28-55) and pMM025 (Δ86-117) are relatively low and are calculated at 54 and 36% for pMM67 mRNAdp, respectively. This result implies that the modification efficiency of mRNA plays an important role in the regulation of cspA expression, in particular the base +118 to +143 region of the 5'-UTR plays an important role in the modification efficiency.

Comparison of the nucleotide sequences of the 5'-UTR region of cold shock can induce genes, cspA and cspG, and as shown in FIG. 12, the well-conserved of these genes is the 13-base sequence (base of cspA +123 At +135).

The 13-base sequence contains a palindromic sequence located at the top just near the Shine-Dalgarmo sequence and forming a stable second structure (ΔG = -9.5 kcal; see FIGS. 12 and 14). It is also complemented to the base 1023-1035 region of 16S rRNA (FIG. 12). In pMM026 (Δ118-143) mRNA, the top-box region is removed, which may be a major cause of poor transformation efficiency of the mRNA.

In order to characterize the role of the top box in the deformation efficiency, two new configurations are made; In one construct (pKNJ37) the correct 13-base top-box sequence is removed from the wild type cspA-lacZ construct (pMM007) and in another construct (pKNJ38) the 13-base sequence is shown in pKM67 (Mitta et al. , 1997). The lacZ gene in pKM67 is fused at base + 26, indicating that the actual deletion in 5'UTR β-galactosidas is expressed at 37 ° C. without any further induction following cold shock. Note that pMM007 and pMM67 have the exact same insert of cspA in different vectors of pRS414 and pKM005, respectively. The cold shock induction pattern of β-galactosidase activity of these two plasmids (pMM007 and pMM67) is similar (data not shown). The cells are modified with pKNJ37, pMM007, pKNJ38 and pKM67, and cold shock induction of β-galactosidase is examined. The deletion of the top box pKNJ37 significantly lowers β-galactosidas at 37 ° C. as well as upon cooling shock. Contrary to the results, when the top box is inserted on top of the pKM67 13 base of the SD sequence, the structural formula of β-galactosidas is increased by about 20% at 37 ° C. (FIG. 13B). The increase is also maintained with cooling shocks. The results support the concept that the top-box sequence relates to the effective modification of cspA. Particular sequences of 5'-UTRs useful in the present invention include 5'-GCCGAAAGGCACA-3 '(SEQ ID NO: 48), 5'-GCCGAAAGGCUCA-3' (SEQ ID NO; 49) and 5'-GCCGAAAGGCCCA-3 '( SEQ ID NO: 50) (see FIG. 12).

It is currently contemplated that cspA expression is regulated to the level of transcriptional mRNA stability and modification as follows: (i) cspA gene contains a strong promoter that is mounted to the UP element, and transcription of the cspA gene causes any protein synthesis upon cold shock Although not required, it operates at both 37 ° C. and 15 ° C. (fang et al., 1997; goldenberg et al., 1997; Mittta et al., 1997). (Ii) cspA mRNA contains a lower-box (DB) sequence underneath the initial codon, which plays an important role in increasing the number of modifications at low temperatures (Etchegaray and Inouye, unpublished: Mitta et al., 1997), (Iii) cspA mRNA contains a generally long 5′-UTR (Tanabe et al., 1992), which constitutes 159 bases and is very unstable at 37 ° C. (Brandi et al., 1996; Goldenberg et al., 1996; Bae et al., 1997; Fang et al., 1997; Jiang et al., 1997; Goldenbert et al., 1997; Mitta et al., 1997). As soon as a cold shock occurs, cspA mRNA is stabilized. Again, stabilization of mRNA following the cold shock does not require any initial protein synthesis. The +26 to +143 region of the 5'-UTR is deleted from the cspA-lacZ fusion construct and high β-galactosidase activity is formed even at 37 ° C (Mitta et al., 1997). In the 5'-UTR, the putative RNaseE cleavage site is at the top of the SD sequence (Fang et al., 1997). The site is considered for extreme instability of cspA mRNA, as it results in 3-base substitution mutations in this region at 150-fold stabilization of mRNA and allows for high CSpA yield even at 37 ° C. (Fang et al. 1997). Thus, it is clear that the long 5'-UTR of normal cspA mRNA may be unstable at 37 ° C. It is speculated that 5′-UTR produces very unstable cspA mRNA at 37 ° C., just as it can induce cspArk cold shock. cspA mRNA accumulates at a temperature that inhibits replication in stabilized and temperature-sensitive RNaseE mutations, but cspA production is not detected under these conditions (Fang et al., 1997), and another role of stabilization of mRNA is 5'-. It may be in the UTR, so it is worth mentioning.

Attempts were made to demonstrate the role of the long 5'-UTR as a replacement for cspA mRNA in cspA expression. A series of 26-32 base deletion mutations was made that encompassed the entire 5'-UTR. The mutated 5′UTR region is transformed into lacZ at the 13th amino acid residue of CspA following the DB sequence. At 37 ° C., pMM022 (Δ2-27), pMM023 (Δ28-55) and pMM026 (Δ118-143) show β-galactosidase activity similar to wild pMM67, pMM024 (Δ56-86) and pMM025 (Δ86-117) shows β-galactosidase activity 10-fold higher than pMM67, indicating that the +117 region at base + 56 is involved upon suppression of cspA expression at 37 ° C.

Based on β-galactosidase activity after shifting down the temperature, the mutants can be divided into three classes; Β-galactosidase in class I [pMM022 (Δ2 + 27) and pMM024 (Δ56-86)] is induced similarly to pMM67 class, and class II [pMM023 (Δ28-55) and pMM (Δ86). -117) has poor cooling shock induction of β-galactosidas, and β-galactosidase activity is very low before and after cooling shock in class III [pMM026 (Δ118-143). It is worth mentioning that the differences do not depend on the stability or amount of mRNA as evidence of FIGS. 10 and 11. This supports the concept that 5'-UTR plays another role in addition to the stability of mRNA, as mentioned above.

The relative strain efficiency after shifting down the temperature for different configurations surprisingly shows a different difference (Fig. 11 C), consistent with the classification of the configurations at 15 ° C as mentioned above. Modification efficiencies of pMM022 (Δ2-27) and pMM024 (Δ56-86) (Class I) are slightly better than wild type pMM67, and modifications of pMM023 (Δ28-55) and pMM025 (Δ86-117) (Class II) The efficiency is 40-50% of pMM67, and the strain efficiency of pMM026 (Δ118-143) (Class III) is 10% or less of pMM67.

In pMM026 (Δ118-143), deletion mutations certainly affect not only stabilization of mRNA but also modification efficiency. The deleted region was found to contain well-conserved 13-base sequences (bases 123- + 135) in mRNA despite cold shocks that can induce the cspA group, cspA, cspG, and cspI (see Figure 12). . The designated sequence top box can form a separate second configuration in both wild type pMm67 and class I configurations (pMM022 (Δ2-27) and pMM024 (Δ56-86)) (FIG. 14). Class I configurations show similar transformation efficiencies as wild type. In class II (pMM023 (Δ28-55) and pMM025 (Δ86-117)), which exhibited 50% of wild type modifications, the second structure was dispersed, but the predetermined second structure near the SD sequence was still in contrast to the nocturnal structure. similar. On the contrary, if the upper box area is deleted [pMM026 (Δ118-143); Class III shows that the deformation efficiency is very poor]. The SD region forms a more stable second structure. As such, the recognition of mRNA is prevented by ribosomes causing poor modification efficiency in pMM026 (Δ118-143). The top box can thus serve to stop the formation of a stable second structure directly above the SD sequence, allowing access to highly ribosomes. Optionally, as the top-box sequence is complemented by the 16S rRNA sequence from base 1023 to 1035 (FIG. 12), the top box sequence can be another cis-element, which is the SD sequence and the bottom box (Mitta et al. , 1997) can increase the efficiency of modification by forming a double 16S rRNA. Since the overall composition uses the same site as cspA to fuse to lacZ, the differences observed in strain efficiency are considered at the strain initial level, not at the level of strain extension. The interaction between mRNA and 16S rRNA plays an important role early in the transformation (McCarthy and Brimacombe, 1994). The 1035 region at base 1023 of the 16S rRNA appears near the site where 30S ribosomes interact with the sixth base from the initial condon (Dontsova et al., 1992; Rinke-Appel et al., 1993). In addition, the increased RNase VI sensitivity at G1020, A1021 and A1022 after assembly of 30S ribosomes indicates that sites containing these residues are exposed to the surface (Power et al., 1998).

Consistent with the suggested role of the top box in the modification, the addition of the top box sequence in pMM67 results in about 20% increase in β-galactosidase activity. On the other hand, the correct 13-base top box sequence (pKNJ37 (Δ123-135 in FIG. 13)) results in a 50% reduction in β-galactosidas at 37 ° C. and lowers the induction level upon cooling shock. The second predetermined structure surrounding the SD sequence of pKNJ37 (Δ123-135) is the same as the wild type (FIG. 14).

In the heat shock reaction, the synthesis of the heat shock schima factor, σ ³² , is regulated at the level of modification and the second structure of rpoH mRNA plays an important role in this regulation (Nagi et al., 1991; Yuzawa et al., 1993). . Recently, the second structure of rpoH mRNA has been chemically determined and the probing of the enzyme is evaluated so that the results are in perfect agreement with the predetermined rpoH second structure presented above (Morita et al., 1999). In addition, mutations in rpoH mRNA predicted to reduce mRNA stability indicate increased rpoH expression and vice versa (Morita et al., 1999). The wild type ropH mRNA and 30S ribosomes are used to assess topping and the toeprinting appearance is temperature dependent overall, which means that at 42 ° C the rpoH mRNA second structure is accessible to ribosomes and at 30 ° C without any other factors It indicates that ropH mRNA structure is self-determined for expression (Mirita et al., 1999). Thus, the proposed rpoH mRNA acts as an RNA heat sensor (Morita et al., 1999). Storz (1999) discussed λ phase cIII mRNA (Altuvia et al., 1989), which may be another example of lcrF mRNA and RNA heat detectors from Yersinia pestis (Hoe et al., 1993).

In summary, stabilization of cspA mRNA following cold shock is a prerequisite for CspA production. Stabilization does not require any initial protein synthesis. As in the present invention, it has been found that modification efficiency, which may involve the second structure of the 5'-UTR, particularly surrounding the SD sequence of cspA mRNA, plays an important role in cspA expression in addition to mRNA stabilization. To learn more, recall the molecular mechanism of regulation of cspA expression, the determination of the second structure of cspA mRNA and the relationship between the second structure and the transformation efficiency. In addition to deletion analysis, point mutation analysis reveals molecular analysis information of the structure and function of the 5'-UTR of cspA mRNA.

Example 11 Role of DB in Cooling Shock Induction of cspB, and Effect of Ideal Matching DB

Earlier, we described that the expression of cspB at low temperatures is mainly controlled by mRNA levels (Etchegaray et al., 1996). Here, DB was first shown to make a significant contribution to the strain induction of cspB at low temperatures. E. coli AR 137, pcnB mutations that retain the pBR322 derivative plasmid at low copy (Lopilato et al., 1986) are transformed into a series of cspB-lacZ modified fusions (FIGS. 15A and 15B). 15C shows β-galactosidase activity at various time points after lowering the temperature from 37 to 15 ° C. After 3 hours at 15 ° C., pB13 and pB17 show high levels of β-galactosidase activity, indicating that codon 3 and 13 regions (including DB) play an important role in cspB high expression at low temperatures. The levels of β-galactosidase units in the SD-erasing constructs comprising pB 13sd and pB 17sd indicate that SD is required for induction of cspB. Primer extension analysis (FIG. 15D) shows that the mRNA levels of pB3 and pB17 are nearly identical, whereas the β-galactosidase activity of pB17 is seven times higher than pB3, indicating that the difference in the lower sequence of the mRNA of the initial codon Not only depends on the amount, but also significantly affects the efficiency of mRNA modification. Based on the amount of mRNA measured using phosphorinase and an increase in β-galactosidase activity between 1 and 2 hours after cold shock induction, the likelihood of modification of pB17 is 18 times higher than pB 3 and 6 times higher than pB13. Is measured. This can be explained by translocation to form additional base pairing between cspB-mRNA (pB17) and 16S rRNA as shown in FIG. 15A. This means that a long DB (pB17) is more effective for modification than a short DB (pB13). When the SD sequence is deleted from pB 13, the modification efficiency is lower than pB 3, meaning that both SD and DB are required for full expression of cspB at low temperatures.

mRNA stability acts on cold shock induction of cspA (Brandi et al., 1996; Goldenberg et al., 1997; Fan et al., 1997). However, the dramatic effect of DB on lacZ expression is compelled to account for the difference in mRNA stabilization at 15 ° C between structures with and without DB. 15E shows that the half-life for pB3, pB13, pB13sd and pB17 is 12, 22, 15 and 20 minutes, respectively.

DB has previously been shown to be essential for the production of CspA at low temperatures (Mitta et al., 1997). However, the wild type DB of cspA has 15 or more 10 pairs that can conform to 16S rRNA anti-DB (Mitta et al., 1997). Thus, when lacZ expression is increased at 15 ° C., 12 (pJJG78DB1) or 15 (pJJG78DB2) bases complement the anti-DB of 16S rRNA at the site after investigating the fifth codon of lacZ under the pJJG78 (see FIG. 15A) cspA control system. Add DB. Mid-log phase cells (pcnB) grown at 37 ° C. were shifted to 15 ° C. and β-galactosidase activity was measured at 1,2 and 3 hours after migration. FIG. 15B shows that β-galactosidas activity was 3 and 8 times higher than pJJG78DB1 and pJJG78DB2, respectively, at 15 ° C. for 1 hour. After 2 and 3 hours at 15 ° C., β-galactosidase activity is increased 3.5-fold and 10.5-fold in pJJG78DB1 and pJJG78G2 than pJJG78, respectively. In addition, the DB effect observed at 37 ° C. was 2 and 4 times higher in β-galactosidase activity of pJJG78DB1 and pJJG78DB2 compared to pJJG78. The amount of lacZ mRNA (FIG. 15C) as well as the stability of the mRNA does not significantly affect between these constructs. The lacZ mRNA half-life from pJJG78m pJJ78D1 and pJJG78DB2 was measured at 27, 23, and 25 minutes, respectively. In addition, computer analysis showed that there was no significant difference in mRNA second structure between pJJG78, pJJ78DB1 and pJJ78DB2, which means that the insertion of an appropriate matched DB into the mRNA second structure could allow for differences in β-galactosidase expression. It means no special effect. The results in between mean that the DB functions as a modification enhancer and that the large complement of anti-DB improves the modification efficiency and that special base-compositions such as the first nucleotide of the DB from pJJG78DB2 can play an important role in DB activity.

Example 12 DB Function Analysis

The experiment was conducted at 15 ° C. To test if the DB operates at 37 ° C., the cspA cooling shock control region of SD of pJJG78 and pJJ78DB2 is replaced by a lac promoter-operator fragment using the lpp promoter and the pINIII vector (Inouy, M. 1983), respectively, pINZ and pINZDBI (FIG. 17), cells (pcnB) are transformed into pINZ or pINZDB1, which exhibits low β-galactosidase activity without the IPTG, the inducer of the lac promoter (FIG. 1C, time 0). Following addition of 1 mM IPTG, β-galactosidase activity is induced in both cells. After 3 hours of induction, the activity of β-galactosidase is increased by 18 and 37 fold over pINZ and pINSDB1, respectively (FIG. 17). However, β-galactosidase activity levels show a dramatic difference between two; Activity with DB (pINZDB1) is 34 times higher than without DB (pINZ), which means that the DB operates at 37 ° C.

The specific activity of β-galactosidas formed from the vectors pINZ and pINZDB1 is about the same (data not shown). The addition of the 5 amino acids present in the β-galactosidase sequence of pINZDB1 (due to DB) adds to the activity of the enzyme. Does not affect In addition, stabilization of β-galactosidase from pINZ and pINZDB1 is equal to half life of about 3 hours (data not shown).

Next, we examined whether the DB function is independent of SD by initial ribosome binding. For this purpose, the SD sequences of pINZDB1, GAGG were changed to GCCC and pINZDB2 (FIGS. 17A and 17B) were obtained. β-galactosidase level of pINZDB2 induction was reduced to 1/7 of pINZDB1, but still three times higher than pINZ at 3 hours after IPTG induction (FIG. 17). However, since pINZDB2 has a second AUG codon, the lower 6 codons may result in the DB insert acting as a second initial codon for the lacZ gene. Instead, N-terminal sequence analysis shows that 100% of β-galactosidase generated from pINZDB2 begins in the second AUG codon and 90% starts in the first AUG codon compared to pINZDB1. In addition, the second AUG codon is processed by translocation but the poor SD sequence (AAGG) in the region corresponds to the second and third codons (FIG. 17B; underlined). In fact, when the second SD was removed from deletion of the 15-base sequence (codons 1-5; pINZDB3 in FIG. 17B), β-galactosidase activity was reduced to background levels at all time points (FIG. 17C). This means that the second SD plays an important role in the modification of pINZDB2 lacZ mRNA (FIG. 17C). It is important that the DB sequence starting at the first AUG codon is cleared at pINZDB4 (FIG. 17B). Thus, high expression of β-galactosidase of pINZDB1 and pINZDB4 is preferably according to the matching DB sequence (FIG. 17B). This result means that (a) the DB only functions when there is a DS, and (b) the location of the DB is elastically initiated at codons 1 or 6.

Example 13: Increase of Deformation by DB

Β-galactosidase activity shown in FIG. 17C indicates that the DB increases the modification of pINZDB1. Therefore, the ratio of β-galactosidase synthesis from pINZ and pINZDB1 was analyzed to effectively test the DB with modification efficiency. The ratio of β-galactosidase synthesis was measured by pulse-labeling cells for 5 minutes with [35S] -methionine using cell harboring pINZ and pINZDB1 after addition of IPTG. After SDS-PAGE, the amount of radioactive β-galactosidase is measured using phosphorimer (FIG. 17D). Before adding IPTG, the β-galactosidase synthesis ratio from pINZ and pINZDB1 is the same. However, with IPTG induction, the ratio of β-galactosidase synthesis from pINZDB1 increased continuously at each time point, resulting in little effect of β-galactosidase synthesis from pINZ. The β-galactosidase synthesis ratio from pINZDB1 4 hours after IPGT addition is 6.5 times higher than pINZ. This result implies that the strain efficiency of pINZDB1 increases as DB is reflected in the increase in the synthesis of β-galactosidas.

To test if the DB increases modification initiation, the performance of lacZ mRNA from pINZ and pINZDB1 was analyzed to form the next polysome. For this experiment, pcnB cells are used to amplify the DB effect. Cells with pINZDB1 cannot form individuals in the LB plate in the presence of 1 mM IPTG, and cells with pINZ form individuals. The lethal effect of IPTG on cells with pINZDB1 is presumably due to overexpression of β-galactosidas. After addition of IPTG, cell growth was stopped by addition of chloramphenicol (0.1 mg / ml) at 15, 30 and 60 minutes and the examined polysome profile is shown in FIG. 17D. From each gradient fragment (500 ml), 200 ml were stopped in nitrocellulose and the amount of lacZ mRNA was analyzed using 24-base antisense oligonucleotides (M13-47 oligonucleotides). The amount of lacZ mRNA was measured by polypolimer and shown in FIG. 17D. Polysomal profiles are similar, but there are significant differences in the distribution of lacZ mRNAs; Primarily lacZ mRNA is located in the upper half of the gradient with pINZ at 15 minutes (fragments 8-14, corresponding to 70S-30S ribosomes), with pINZDB1 the main peak (fragments 3-8) formed in the lower half of the gradient do. At 30 minutes, lacZ mRNA from pINZ is shifted to the position of 70s ribosomes and lacZ mRNA from pINZDB1 remains in the same pattern as at 15 minutes. At 60 minutes, major fragments of lacZ mRNA remain in the upper half of the gradient from pINZ, and lacZ mRNA from pINZDB1 is widely distributed from higher polysomes as 70S ribosomal fragments. Thus, cell harboring pINZDB1 reduces the defects of free ribosomes as a result of large expression of highly deformable DB-containing mRNA (vind et al., 1993), which is why individuals do not form on LB plates containing 1 mM IPTG. May be present. This result means that the DB preferably increases the efficiency of polysome formation with increasing initial strain.

To determine the exact effect of the DB from the experiment, the lacZ mRNA amount and β-galactosidase activity were measured at the same time points taken in the polysome profile (15,30 and 60 minutes after IPTG induction). As shown in FIG. 18A, the amount of lacZ mRNA reaches nearly maximum levels at 15 minutes for pINZ and pINZDB1. The phosphoriner mergery analysis of the results indicated that the amount of pINZDB1 mRNA was 1.5, 1.4 and 1.3 times higher than that of pINZ mRNA. High mRNA levels for pINZDB1 preferably contribute to the high effect polysome formation of pINZDB1 that can stabilize mRNA (Iost and Dreyfus 1995). Induction of β-galactosidase activity is shown in FIG. 18B. In the case of pINZDB1, the activity is very high in the absence of IPTG and increases to 18,500 to 64,000 (3.5-fold) after a 2.5 hour incubation with the addition of IPTG. In the case of pINZ, the background activity before IPTG induction is very low, increasing to 900 to 2,900 units (3x) at 2.5 hours. The increase in β-galactosidase activity of pINZDB1 between 30 and 60 minutes is 35 times higher than pINZ, so the efficiency of β-galactosidase production for pINZDB1 is 26 times higher for pINZdp based on the amount of mRNA. Is measured. Thus, the high rate of β-galactosidas production from pINZDB1 is due to the high efficiency of polysome formation.

Next, β-galactosidase was tested in a cell-free system using pINZ and pINZDB1 to account for the strain-increasing effect of DB. [ ³⁵ S] methionine incorporation into β-galactosidas (band G) with pINZDB1 (lane 2, FIG. 19) is eight times higher than pINZ (lane 1), and β-galactosidas (band L) production is It is almost the same in both lanes.

Absence of ribosomal protein S2 resolves the anti-DB sequence from the stem of the second syllable

Claims (56)

An isolated nucleic acid molecule that sustains expression of a cold shock inducing gene under conditions that produce a cold shock response in bacteria. The isolated nucleic acid molecule of claim 1, wherein the molecule comprises a 5′-UTR or the same sequence of the cold shock inducing gene. The isolated nucleic acid molecule of claim 2, wherein the 5′-UTR is a 5′-UTR of a cold shock inducing gene selected from the group consisting of cspA, cspB and csdA. The isolated nucleic acid molecule of claim 2, wherein the 5′-UTR consists of a cold shock inducing gene box or a sequence equivalent thereto. The isolated nucleic acid molecule of claim 3, wherein the 5′-UTR consists of +1 to +11 nucleotides or equivalent sequences thereof with cspA 5′-UTR. The isolated nucleic acid of claim 1, wherein the cold shock inducing gene interacts with the CspA protein. An isolated nucleic acid molecule that inhibits the expression of a cold shock inducing gene under physiological conditions. 8. The isolated nucleic acid molecule of claim 7 comprising at least a portion of the 5'-UTR of the cold shock inducing gene. The isolated nucleic acid molecule of claim 8, wherein the cold shock inducing gene is selected from the group consisting of cspA, cspB and csdA. The isolated nucleic acid molecule of claim 8, wherein the cold shock inducing gene consists of nucleotides equal to +56 to +117 nucleotides of cspA or to +56 to +117 nucleotides of cspA. An unencoded nucleic acid molecule that enhances translation of a cold shock inducing gene under conditions that cause a cold shock response in bacteria. 12. The unencoded nucleic acid molecule of claim 11 comprising at least a portion of the 5'UTR of the cold shock inducing gene. The isolated nucleic acid molecule of claim 12, wherein the cold shock inducing gene is selected from the group consisting of cspA, cspB and csdA. 15. The isolated nucleic acid molecule of claim 13, wherein the cold shock inducing gene consists of nucleotides equal to +123 to +135 nucleotides of cspA or to +123 to +135 nucleotides of cspA. 15. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid molecule has a sequence selected from the group consisting of the 48th, 49th, or 50th sequences. A nucleic acid vector consisting of a nucleic acid molecule and a downstream box that enhance the translation of a cold shock inducing gene under conditions causing a cold shock reaction in bacteria. The nucleic acid vector of claim 16 comprising a shine-dalgano sequence. For nucleic acid vectors, at least a portion of the vector comprises a cold shock inducing gene box, a translation enhancer and a downstream box, wherein the vector sustains gene expression and enhances translation under conditions that cause a cold shock response in bacteria. A nucleic acid vector characterized by. In nucleic acid vectors, gene expression is maintained under physiological stress conditions that cause cold shock reactions in bacteria, enhanced translation, inhibition of gene expression under physiological conditions, cold shock induced gene boxes, At least a portion of the 5'-UTR of the cold shock inducing gene to inhibit expression, at least a portion of the 5'-UTR of the cold shock inducing gene to enhance translation of the cold shock inducing gene, and a downstream box Nucleic Acid Vectors. The vector of claim 16, wherein the gene is a cold shock inducing gene. 19. The vector of claim 18, wherein the gene is a cold shock inducing gene. 20. The vector of claim 19, wherein the gene is a cold shock inducing gene. The vector of claim 16, wherein the gene is a heterologous gene. 19. The vector of claim 18, wherein said gene is a heterologous gene. The vector of claim 19, wherein the gene is a heterologous gene. The vector of claim 16, comprising a promoter, one or more restriction sites located downstream of the 5′-UTR, and a downstream box into which the DNA fragment can further be inserted. The vector of claim 18, comprising a promoter, one or more restriction sites located downstream of the 5′-UTR, and a downstream box into which the DNA fragment can further be inserted. 20. The vector of claim 19 comprising a promoter, one or more restriction sites located downstream of the 5'-UTR, and a downstream box into which the DNA fragment can be further inserted. 27. The vector of claim 26, wherein said additional DNA fragment encodes a cold shock inducing gene. The vector of claim 27, wherein said additional DNA fragment encodes a cold shock inducing gene. 29. The vector of claim 28, wherein said additional DNA fragment encodes a cold shock inducing gene. 27. The vector of claim 26, wherein said additional DNA fragment encodes a heterologous gene. The vector of claim 27, wherein said additional DNA fragment encodes a heterologous gene. 29. The vector of claim 28, wherein said additional DNA fragment encodes a heterologous gene. A transformed bacterium containing the vector according to claim 16. A transformed bacterium containing the vector according to claim 18. A transformed bacterium containing the vector according to claim 19. In a method of overexpressing a gene, Transforming the bacteria with the nucleic acid vector according to claim 16; And Placing the bacteria under conditions that produce a cold shock reaction Overexpression method of the gene comprising a. In a method of overexpressing a gene, Transforming the bacteria with the nucleic acid vector according to claim 18; And Placing the bacteria under conditions that produce a cold shock reaction Overexpression method of the gene comprising a. In a method of overexpressing a gene, Transforming the bacteria with the nucleic acid vector according to claim 19; And Placing the bacteria under conditions that produce a cold shock reaction Overexpression method of the gene comprising a. 39. The method of overexpression of a gene according to claim 38, wherein said overexpression decreases the expression of one or more endogenous proteins. 40. The method of overexpressing a gene according to claim 39, wherein said overexpression decreases the expression of one or more endogenous proteins. 41. The method of overexpressing a gene according to claim 40, wherein said overexpression decreases the expression of one or more endogenous proteins. 39. The method of over-expression of a gene according to claim 38, wherein the conditions for generating a cold shock response include placing the bacteria at a low temperature at which a cold shock response can be induced. 40. The method of overexpressing a gene according to claim 39, wherein the conditions for generating a cold shock reaction include placing the bacteria at a low temperature at which a cold shock response can be induced. 39. The method of over-expression of a gene according to claim 38, wherein the conditions for generating a cold shock response include placing the bacteria at a low temperature at which a cold shock response can be induced. 45. The method of overexpressing a gene according to claim 44, wherein said low temperature is 10-15 [deg.] C. 46. The method of overexpressing a gene according to claim 45, wherein said low temperature is 10 to 15 ° C. 47. The method of overexpression of a gene according to claim 46, wherein said low temperature is 10-15 [deg.] C. Heterologous genes can be expressed at physiological temperature under cold shock inducing conditions, and include promoters; At least a portion of the 5'-UTR of the cold shock protein gene; Shine-dalgano sequence; Start translation codons; Downstream boxes of cold shock inducing genes; And at least one restriction enzyme recognition site for recognition of the heterologous gene. 51. The vector of claim 50 comprising an additional nucleic acid fragment encoding a cold shock inducing gene. 51. The vector of claim 50 comprising an additional nucleic acid fragment encoding a heterologous gene. A transformed bacterium comprising the vector according to claim 50. In a method of overexpressing a gene, Transforming the bacteria with the nucleic acid vector according to claim 50; And Placing the bacteria under conditions that produce a cold shock reaction Overexpression method of the gene comprising a. 55. The method of claim 54, wherein the condition causing the cold shock response comprises placing the bacteria at a low temperature at which a cold shock response can be induced. 46. The method of overexpressing a gene according to claim 45, wherein said low temperature is 10 to 15 ° C.