EP0914424A1 - Neuartige gene auf chromosom 16, zusammensetzungen, ihre herstellung und verwendung - Google Patents

Neuartige gene auf chromosom 16, zusammensetzungen, ihre herstellung und verwendung

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Publication number
EP0914424A1
EP0914424A1 EP97903844A EP97903844A EP0914424A1 EP 0914424 A1 EP0914424 A1 EP 0914424A1 EP 97903844 A EP97903844 A EP 97903844A EP 97903844 A EP97903844 A EP 97903844A EP 0914424 A1 EP0914424 A1 EP 0914424A1
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EP
European Patent Office
Prior art keywords
leu
ala
gly
ser
val
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP97903844A
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English (en)
French (fr)
Inventor
Gregory M. Landes
Timothy C. Burn
Timothy D. Connors
William R. Dackowski
Terence J. Van Raay
Katherine W. Klinger
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Genzyme Corp
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Genzyme Corp
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Priority claimed from US08/665,259 external-priority patent/US6028173A/en
Priority claimed from US08/762,500 external-priority patent/US6030806A/en
Application filed by Genzyme Corp filed Critical Genzyme Corp
Publication of EP0914424A1 publication Critical patent/EP0914424A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • exon trapping is that the expression of cloned genomic DNAs (cosmid, PI or YAC) is driven by a heterologous promoter in tissue culture cells. This allows for coding sequences to be identified without prior knowledge of their tissue distribution or developmental stage of expression.
  • a second advantage of exon trapping is that exon trapping allows for the identification of coding sequences from only the cloned template of interest, which eliminates the risk of characterizing highly conserved transcripts from duplicated loci. This is not the case for either cDNA selection or direct library screening.
  • Exon trapping has been used successfully to identify transcribed sequences in the Huntington' s disease locus (Ambrose et al . , Hum . Mol . Genet . 1:697-703, 1992; Taylor et al . , Nature Genet . 2:223-227, 1992; Duyao et al . , Hum . Mol . Genet . 2:673-676, 1993) and BRCA1 locus (Brody et al . , Genomics 25:238-247 , 1995; Brown et al . , Proc . Natl . Acad. Sci . , USA 92:4362-4366, 1995) .
  • This chromosomal segment serves as a challenging test for large-insert cloning systems in E. coli and yeast since it resides in a GC-rich isochore (Saccone et al . , Proc . Natl . Acad. Sci . , USA 89:4913-4917, 1992) with an abundance of CpG islands (Harris et al . , Genomics 7:195- 206, 1990; Germino et al . , supra . 1992) , genes (Germino et al . , supra . 1993) and Alu repetitive sequences (Korenberg et al . , Cell 53:391-400, 1988) .
  • Chromosome 16 also contains more low-copy repeats than other chromosomes with almost 25% of its cosmid contigs hybridizing to more than one chromosomal location when analyzed by fluorescence in si tu hybridization (FISH) (Okumura et al . , Cytogenet . Cell Genet . 67:61-67, 1994) .
  • FISH fluorescence in si tu hybridization
  • isolated nucleic acids encoding a human netrin, a human ATP binding cassette transporter, a human ribosomal L3 subtype, and a human augmenter of liver regeneration.
  • the present invention further provides isolated protein products encoded by a human netrin gene, a human ATP binding cassette transporter gene, a human ribosomal L3 gene, and a human augmenter of liver regeneration gene.
  • the present invention provides nucleic acid probes that hybridize to invention nucleic acids as well as isolated nucleic acids comprising unique gene sequences located on chromosome 16.
  • vectors containing invention nucleic acids as well as host cells transformed with invention vectors.
  • Transgenic non-human mammals that express invention polypeptides are provided by the present invention.
  • the present invention includes antisense oligonucleotides, antibodies and compositions containing same.
  • the invention provides methods for identifying compounds that bind to invention polypeptides. Such compounds are useful for modulating the activity of invention polypeptides.
  • Figures 4A and 4B show 1743 bp of hNET cDNA and deduced amino acid sequence coding for a human homologue of chicken netrin genes (SEQ ID NOs:20 and 21) .
  • Figures 4C and 4D show the nucleotide sequence of the 1.9 kb hNET cDNA including both 5' and 3' UTRs (SEQ ID NO:78) .
  • Figure 5 shows an amino acid comparison between chicken netrin-1 (SEQ ID NO:22), chicken netrin-2 (SEQ ID NO:23) and hNET (SEQ ID NO:21) . Shaded boxes denote regions of identical homology.
  • the laminin domains V and VI and the C-terminal domain (C) are indicated by arrows with domain V divided into three sub-components (V-1 to V- 3) .
  • the asterisks identify a motif for adhesion/signaling receptors.
  • Figure 6 shows a graphical representation of the homology between domains of chicken netrin-1, chicken netrin-2 and hNET.
  • Figure 10 shows the region of the transcriptional map of the PKDl locus from which Pi clones 49.10D, 109.8C and 47.2H were isolated.
  • the open boxes represent trapped exons with their relative position indicated below the RPL3L (SEM L3) gene.
  • c, r and h identify the location of the capture, repair and hybridization oligonucleotides, respectively.
  • FIGS 11A-11B show the nucleotide and deduced amino acid sequence of the SEM L3 cDNA, now designated RPL3L (SEQ ID NOs:28 and 29) .
  • the 5' upstream inframe stop codon is underlined and the arrows indicate the site of the polyA tract of the two shorter cDNA clones that were also isolated.
  • Figure 12 shows a comparison of the deduced amino acid sequences from human (SEQ ID NO:30), bovine (SEQ ID NO:31) , murine (SEQ ID NO:32) and the RPL3L (SEM L3) (SEQ ID NO:29) genes. Dashes indicate sequence identity to the human L3 gene. The nuclear targeting sequence at the N-terminal end is shaded and the bipartite motif is boxed.
  • Figures 15A-15J show the nucleotide and deduced amino acid sequence of full-length hABC3 cDNA (SEQ ID NOs:74 and 75) .
  • Figure 16 shows a physical map of the region containing the hABC3 gene.
  • FIG 17B shows a schematic diagram of the ABC3 protein showing the transmembrane (TM) domains, ATP binding cassette (ABC) domains, Linker and HHl domains.
  • cDNA complementary DNA
  • a "contig” is a continuous stretch of DNA or DNA sequence, which may be represented by multiple, overlapping, clones or sequences.
  • a "cosmid” is a DNA plasmid that can replicate in bacterial cells and that accommodates large DNA inserts from about 30 to about 51 kb in length.
  • exon trapping refers to a method for isolating genomic DNA sequences that are flanked by donor and acceptor splice sites for RNA processing.
  • Amplification of DNA denotes a reaction that serves to increase the concentration of a particular DNA sequence within a mixture ot DNA sequences. Amplification may be carried out using polymerase chain reaction (PCR) (Saiki et al . , Science, 239:487, 1988), ligase chain reaction (LCR) , nucleic acid- specific based amplification (NSBA) , or any method known in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NBA nucleic acid- specific based amplification
  • RT-PCR refers to coupled reverse transcription and polymerase chain reaction. This method of amplification uses an initial step in which a specific oligonucleotide, oligo dT, or a mixture of random primers is used to prime reverse transcription of RNA into single-stranded cDNA; this cDNA is then amplified using standard amplification techniques e.g. PCR.
  • Exon trapping was performed using an improved trapping vector (Burn et al . , Gene 161:183-187, 1995), with the resulting exon traps being characterized by DNA sequence analysis.
  • exon traps were compared to the cDNA sequences for those genes known to be in the interval around the PKDl gene ( Figure 1) .
  • Single exon traps were obtained from the human homologue of the ERV1 (Lisowsky et al . , Genomics 29:690-697, 1995) and the ATP6C proton pump genes (Gillespie et al . , Proc . Natl . Acad. Sci .
  • the horizontal line at the top of Figure 1 shows the position of relevant DNA markers with the scale (in kilobases) .
  • the position of NotI sites is shown below the horizontal line.
  • the position and orientation of the known genes is indicated by arrows with the number of exon traps obtained from each gene shown in parentheses.
  • the position of the transcription units described in this report (A through M) are shown below the known genes.
  • the Genbank Accession numbers of corresponding exon traps are shown below each transcriptional unit.
  • PI clones are indicated by the overlapping lines with the name of the clone shown above the line.
  • the position of trapped exons which did not map to characterized transcripts are shown below the Pi contig. Vertical lines denote the interval within the PI clone(s) detected by the exon traps in hybridization studies.
  • the m ⁇ AP3 protein a zinc finger-containing transcription factor, is believed to function as a negative regulator for genes encoding proteins responsible for the inhibition of cell cycling (Fognani et al . , supra . ) .
  • the two exon traps were linked by PCR, with the resulting 1.2 kb PCR product being 85% identical at the nucleotide level to the murine ⁇ AP3 cDNA.
  • exon trap L48741 SEQ ID N0:1
  • N-acetylglucosamine-6-phosphate deacetylase from C. Elegan ⁇ (SEQ ID N0:2), E. coli (SEQ ID N0:3) and Haemophilu ⁇ (SEQ ID NO:4) .
  • the EGF repeat from netrin-1 (SEQ ID NO:7) , netrin-2 (SEQ ID NO:6) and UNC-6 (SEQ ID NO:8) are shown aligned to one of the translated netrin-like exon traps (Genbank Accession No. L75917) (SEQ ID N0:5) .
  • An alignment of sequences from the second netrin-like exon trap Genbank Accession No.
  • cDNA library screening and PCR based approaches were used to clone transcribed sequences containing selected exon traps.
  • RT-PCR was used to link individual exon traps together in cases where the two exon traps had homology to similar sequences in the databases .
  • 3 ' RACE or cDNA library screening was used to obtain additional sequences. Sequences from the exon traps and cloned products were used to map the position, and when possible the orientation, of the corresponding transcription units.
  • cDNAs were isolated using sequences derived from a separate 94.10H exon trap (Genbank Accession No. L48738) and the position and orientation of the corresponding transcription unit were determined. Two cDNA species were obtained using exon trap L48738 as a probe, with the only homology between the two species arising from the 109 bases contained in the exon trap. Using oligonucleotide probes, the transcription unit was mapped to a position near the 26-6DIS DNA marker, in a telomeric to centromeric orientation; however, only one of the cDNA species mapped to the PI contig (transcript B in Figure 1) .
  • the second cDNA species originated from a region outside of the PI contig, possibly from the duplicated 26-6PROX marker located further centromeric in 16pl3.3 (Gillespie et al . , Nuc . Acids Res . 18:7071-7075, 1990) .
  • the 110.IF Pi clone contains at least two genes in addition to the ATP6C gene.
  • BLASTX N-acetylglucosamine-6-phosphate deacetylase
  • Exon trap L75917 Sequences encoded by exon trap, L75917, were shown to have significant homology with the C-terminal most epidermal growth factor (EGF) repeat found in the netrin and UNC-6 proteins ( Figures 2 and 20A) .
  • Exon trap L75917 encodes sequences which are 98% identical to sequences from the third epidermal growth factor (EGF) repeat of chicken netrin-2 and 90% identical to sequences from the same region of netrin-1.
  • the netrin-like trap, L75916 encodes sequences from the more divergent C-terminal domain of the netrins which are 43% identical to sequences contained in the C-terminal domain of netrin-1 and netrin-2 ( Figures 2 and 20A) .
  • Chicken netrin-1 and netrin-2 have been shown to function as chemoattractants for developing spinal commissural axons (Serafini et al . , Cell 78:409-424, 1994; Kennedy et al . , Cell 78:425-435, 1994) with netrin-1 also acting as a chemorepellant for trochlear motor axons (Colamarino and Tessier-Lavigne, Cell 81:621-629, 1995) . Comparative analysis revealed the presence of extensive homology between the chicken netrins and C. elegans UNC-6 protein which is required for circumferential cell migration and axon guidance (Hedgecock et al .
  • the genomic interval containing the netrin-like exon traps was sequenced in order to obtain additional sequence information from the gene and to rule out the possibility that the exon traps were derived from a pseudogene.
  • the netrin-like exon traps were mapped to a 6 kb Xhol fragment. See, for example, Figure 18 wherein relevant DNA markers are shown on top of the horizontal line, with Notl sites (N) being shown below the line.
  • N Notl sites
  • Additional sequence was obtained using internal primers as well as end sequence from the parental Xhol fragments.
  • a total of 6.8 kb of genomic sequence with an overall redundancy of 7-fold was sequenced.
  • the GC-content for the sequenced region was found to be 68.9%, which is slightly higher than the 62.8% observed for the 53 kb of genomic sequence from the PKDl gene, located 350 kb further telomeric (The American PKDl Consortium, 1995, supra ; Burn et al . , 1996, supra) .
  • GRAIL2 analysis predicted six exons within the 6.8 kb of genomic sequence with database analysis indicating that all but one exon (exon 1) , encoded sequences with homology to the chicken netrins .
  • Figure 19A shows a GRAIL2 analysis of coding sequences in the 6.8 kb of genomic sequence from the 53.8B PI, with the gray scale denoting GC-content (white to light gray is GC rich and gray to black is AT rich) , vertical boxes indicating relative quality of the predicted exons .
  • a graphical depiction of the predicted exons is shown above the vertical boxes with light colored boxes denoting exons with a score of "excellent” ( >80% probability) and dark colored boxes denoting exons with a score of "good” (>60% probability) .
  • the position of exon traps L75917 and L75916 are shown above the GRAIL2 predicted exons.
  • the structure of the gene based on comparison of the RT-PCR products and genomic sequence is shown at the top, the position of the exons in the genomic sequence is shown by the numbers above the exons. The 5' and 3' untranslated regions are also shown.
  • genomic sequence was compared to the protein sequences of the chicken netrins using a Pustell DNA/protein matrix.
  • the genomic sequence (translated in all six frames) was compared to chicken netrin-2 in Figure 19B, using a PAM250 matrix with the minimum homology set at 50% and the window set at 20. Regions of homology are shown by heavy diagonal lines. Five exons were predicted by this analysis, with only the first GRAIL2 predicted exon not appearing to be bona fide . Sequences from the two exon traps were also predicted by GRAIL2; however, there were noteworthy differences ( cf Figure 19A) .
  • GRAIL2 included an additional 55 bp at the 5' end of the exon.
  • the first of the two exons present in exon trap L75916 was not predicted by GRAIL2, while GRAIL2 added additional bases to the 5 ' and 3 ' ends of the second exon present in this exon trap.
  • EST Expressed Sequence Tags
  • This 477 bp contiguous sequence aligns to the 5 ' end of the human netrin cDNA and includes 47 bp of 5 ' UTR and sequences encoding the N-terminal 143 amino acids.
  • a comparison of the deduced human and murine protein sequence indicated that the two proteins were 89.5% (128/143) identical.
  • RT-PCR was performed using primers designed from the predicted exons. Since the predicted human netrin appeared to slightly more homologous to netrin-2 than netrin-1 (57% versus 54%, respectively) and netrin-2 is expressed in the spinal cord of chicken, adult human spinal cord polyA+ RNA was utilized as a template. RT-PCR products were obtained with only a portion of the primer pairs; however, even this required the use of nested primers and two rounds of PCR, with low yields making it necessary to use hybridization and radiolabeled probes to visualize the products. The low yield, and lack of RT-PCR products in some cases, was attributed to the high GC-content of the products (70-80%) .
  • the human netrin protein is predicted to be 580 amino acids in size, with the common domain structure of the netrin family being conserved.
  • positions where the chicken netrins and UNC-6 sequences match the human sequence are denoted by periods while gaps introduced during the alignment are shown by hyphens. Arrows above the sequence alignment show the boundaries of the laminin VI and V domains, and C-terminal region (C) as described (Serafini et al . , Cell 78: 409-424, 1994) .
  • the signal sequence (S) is also shown.
  • V-1, V-2, and V-3 designate each of the EGF domains that constitute domain V.
  • FIGS 4A and 4B The hNET coding sequence and its predicted protein product are shown in Figures 4A and 4B.
  • Figures 4C and 4D show full length hNET cDNA including both 5' and 3' UTR sequence.
  • Human netrins may have a significant role in neural regeneration. Though netrins do not by themselves promote axon growth, they do play a role in the orientation of axon growth. The combination of growth promoting activities with axon guidance cues would be a necessary requisite for directed neural regeneration.
  • Exon trapping results further show that there is a novel ATP Binding Cassette (ABC) transporter in the PKDl locus located between the LCN1 and D16S291 markers in a centromeric to telomeric orientation.
  • ABC ATP Binding Cassette
  • Database searches with the exon trap sequences show homology to the murine ABCl and ABC2 genes (Luciani et al . , supra . 1994) .
  • the human homologs of murine ABCl and ABC2 have been cloned and mapped to human chromosome 9 (Luciani et al . supra . 1994) .
  • Sequences derived from the trapped exons along with those from cDNA selection and SAmple SEquencing (SASE) were used to recover overlapping partial cDNA clones.
  • H C H m a Gene as denoted in Figure 1.
  • oligonucleotide was used as a sense primer.
  • r m d Sequence of oligonucleotides. In the Genetrapper experiments, this oligonucleotide was used in the repair step. For 3 'RACE to experiments,- this was the internal primer. For RT-PCR experiments, this was the anitsense primer. Size of clone obtained using the primer pair.
  • oligonucleotide used to obtain additional sequences.
  • this oligonucleotide was used in the diiect selection step.
  • the designated oligonucleotide was used as a sense primer.
  • a 1.1 kb RT-PCR product which links the three exon traps from transcript F, with the RT-PCR product detecting a 7 kb message on Northern blots has been obtained. Based on a search of the dbEST database, a cDNA from this region was obtained with sequences from exon traps L75924 and L75925 being contained in cDNA 49233 from the I.M.A.G.E. Consortium (Lennon et al . , supra . ) . The presence of both cloned reagents in the same transcription unit has been confirmed using RT-PCR.
  • the ATP binding cassette (ABC) transporters comprise a family of more than 100 proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells (Higgins, C. F., Annu . Rev. Cell . Biol . 8:67-113, 1992; Higgins, C. F. Cell 82:693-696, 1995) . Proteins belonging to the ABC transporter superfamily are linked by strong structural similarities . Typically ABC transporters have four conserved domains, two hydrophobic domains which may impart substrate specificity (Payne et al . , Mol . Gen . Genet . 200:493-496, 1985; Foote et al .
  • ABC transporters exist either as single large symmetrical proteins containing all four domains or as dimers resulting from the association of two smaller polypeptides each containing a hydrophobic and ATP-binding domain. Examples of this multimeric structural form are human TAP proteins (Kelly et al . , Nature 355:641- 644 1992) and the functional PMP70 protein (Kamijo et al . , J. Biol . Chem . 265:4534-40 1990) . This multimeric structure is also found in numerous prokaryotic ABC transporters.
  • the hydrophobic regions are comprised of up to six transmembrane spanning segments.
  • Each ATP binding domain operates independently and may or may not be functionally equivalent (Kerem et al . , Science 245:1073-80 1989; Mimmack et al . , Proc . Na tl . Acad. Sci . , USA 86:8257- 61 1989; Cutting et al . , Nature 346:366-369 1990; Kerppola et al . , J. Biol . Chem. 266:9857-65 1991) .
  • the presence of numerous polar residues and potential phosphorylation sites in the linker domain suggest that this region may play a regulatory role perhaps similar to that of the R-domain of CFTR (Kerem et al . , supra . ) .
  • the four proteins also contain a hydrophobic region, the HHl domain (Luciani et al . , supra . 1994), within the conserved linker domain. Although there is little homology at the sequence level between the HHl domains of hABC3 and the murine ABCs, they appear to be structurally conserved with each domain predicted to have S-sheet conformation.
  • ABCl, ABC2 and hABC3 have different functional roles.
  • the differences present in the transmembrane and linker domains of ABCl, ABC2 and hABC3 may confer each with a unique substrate specificity.
  • alterations and mutations in the transmembrane domains of both prokaryotic and eukaryotic ABC transporters have been shown to alter substrate specificity (Payne et al . , supra . ; Foote et al . , supra . ; Covitz et al . , supra . ) while changes to the R-domain of CFTR have been shown to alter its ion selectivity (Anderson et al . , supra .
  • Murine ABCl and ABC2 have been shown to be expressed at varying levels in a wide variety of adult and embryonic tissues, with the highest levels of ABCl expression being seen in pregnant uterus and regions rich in monocytic cells while highest levels of ABC2 expression were seen in brain (Luciani et al . , supra . 1994; Luciani et al . , supra . 1996) .
  • hABC3 is preferentially expressed in lung with significantly lower levels of expression being seen in brain, heart, and pancreas.
  • ABC transporters have been described for substrates ranging from small ions to large polysaccharides and proteins. Based on the high level of expression in lung, the substrate for hABC3 may play an integral role in the lung function, including ion or polysaccharide transport. Further clues may be provided by a closer examination of hABC3 expression in the lung. These studies would include the identification of the lung cells responsible for hABC3 expression as well as determining the subcellular localization of hABC3. The identification and cloning of the hABC3 cDNA may have implications for cystic fibrosis, since it contains a potential R-domain and is expressed at highest levels in the lung. If hABC3 does play an integral role in lung function, then modulation or alteration of hABC3 substrate specificity could have significant therapeutic implications for CF.
  • the present invention provides a novel human ABC gene which has homology to the murine ABCl and ABC2 genes, as well as sequences predicted to be encoded by cosmid C48B4.4 from C. elegans (Wilson et al . , supra . ) .
  • a 6.4 kb cDNA has been assembled for the hABC3 transporter.
  • the assembled cDNA contains a 5116 nucleotide long open reading frame encoding 1705 amino acids, with the predicted protein having a molecular weight of 191 kDa.
  • the proposed start methionine is 50 bp upstream of the 5 ' end of clone ABCgt.l.
  • the expression pattern of the previously identified human L3 gene and the novel human RPL3L was determined using multiple tissue Northern blots.
  • the human L3 gene showed a ubiquitous pattern of expression in all tissues with the highest expression in the pancreas.
  • the novel gene described herein is strongly expressed in skeletal muscle and heart tissue, with low levels of expression in the pancreas.
  • This novel gene, RPL3L (Ribosomal Protein L3-Like) is located in a gene-rich region near the PKDl and TSC2 genes on chromosome 16pl3.3.
  • transcript M Sequences encoded by transcript M were shown to have homology to pilB from Neisseria gonorrhoeae (Taha et al . , EMBO J. 7:4367-4378, 1988) as well as to a computer predicted 17.2 kDa protein encoded by cosmid F44E2.6 from C. elegans (Wilson et al . , supra . ) .
  • the pilB protein has homology to histidine kinase sensors and has been shown to play a role in the repression of pilin production in Neisseria gonorrhoeae (Taha et al . , supra . 1988; Taha et al . , Mol . Microbiol . 5:137-148, 1991) .
  • residues conserved between pilB, transcript M and the C. elegans, yeast, and Haemophilus sequences do not include the conserved histidine kinase domains from piIB (Taha et al . , supra . 1991) .
  • ALR is a growth factor which augments the growth of damaged liver tissue while having no effect on the resting liver. Studies have demonstrated that rat ALR is capable of augmenting hepatocytic regeneration following hepatectomy.
  • This ALR-like exon trap was also shown to contain sequences from the recently described hERVl gene, which encodes a functional homologue to yeast ERV1 (Lisowsky et al . , supra . ) .
  • a 468 bp cDNA, hALR has been obtained from the human ALR gene ( Figure 13) .
  • the ALR sequences encode a 119 amino acid protein which is 84.8% identical and 94.1% similar to the rat ALR protein ( Figure 14) .
  • human ALR has significant implications in the treatment of degenerative liver diseases.
  • biologically active rat ALR has been produced from COS-7 cells expressing rat ALR cDNA (Hagiya et al. , supra . ) .
  • recombinant hALR could be used in the treatment of damaged liver.
  • a construct expressing hALR could be used in gene therapy to treat chronic liver diseases.
  • the present invention encompasses novel human genes an isolated nucleic acids comprising unique exon sequences from chromosome 16.
  • the sequences described herein provide a valuable resource for transcriptional mapping and create a set of sequence-ready templates for a gene-rich interval responsible for at least two inheritable diseases.
  • the present invention provides isolated nucleic acids encoding human netrin (hNET) , human ATP Binding Cassette transporter (hABC3) , human ribosomal L3 (RPL3L) and human augmenter of liver regeneration (hALR) polypeptides.
  • the present invention further provides isolated nucleic acids comprising unique exon sequences from chromosome 16.
  • nucleic acids also referred to as polynucleotides encompasses RNA as well as single and double-stranded DNA, cDNA and oligonucleotides.
  • isolated means a polynucleotide that is in a form that does not occur in nature.
  • DNA probes derived from the human netrin gene, hNET, the human ABC transporter gene, hABC3, the human ribosomal protein L3 gene, RPL3L, or the human augmenter of liver regeneration gene, hALR are particularly useful for this purpose.
  • DNA and cDNA molecules that encode invention polypeptides can be used to obtain complementary genomic DNA, cDNA or RNA from human, mammalian, or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, by methods described in more detail below.
  • the present invention encompasses isolated nucleic acid sequences, including sense and antisense oligonucleotide sequences, derived from the sequences shown in Figures 3, 4, 8, 11 and 15.
  • hNET-, hABC3-, RPL3L- (SEM L3-) , and hALR-derived sequences may also be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, and the like.
  • the nucleic acids can be modified to alter stability, solubility, binding affinity, and specificity.
  • nucleic acid manipulations use methods that are well known in the art, as disclosed in, for example, Sambrook et al . , Molecular Cloning, A Laboratory Manual 2d Ed. (Cold Spring Harbor, NY, 1989), or Ausubel et al . , Current Protocols in Molecular Biology (Greene Assoc, Wiley Interscience, NY, NY, 1992) .
  • nucleic acids examples include RNA, cDNA, or genomic DNA encoding a human netrin, a human ABC transporter, a human ribosomal L3 subtype, or a human augmenter of liver regeneration polypeptide.
  • Such nucleic acids may have coding sequences substantially the same as the coding sequence shown in Figures 3, 4, 8, 11 and 15, respectively.
  • This invention also encompasses nucleic acids which differ from the nucleic acids shown in Figures 3, 4, 8, 11 and 15, but which have the same phenotype, i.e., encode substantially the same amino acid sequence set forth in Figures 3, 4, 8, 11 and 15, respectively.
  • Phenotypically similar nucleic acids are also referred to as “functionally equivalent nucleic acids”.
  • the phrase "functionally equivalent nucleic acids” encompasses nucleic acids characterized by slight and non- consequential sequence variations that will function in substantially the same manner to produce the same protein product(s) as the nucleic acids disclosed herein.
  • functionally equivalent nucleic acids encode proteins that are the same as those disclosed herein or that have conservative amino acid variations.
  • nucleic acids encoding human netrin, human ABC3 transporter, human ribosomal L3 subtype, and human augmenter of liver regeneration polypeptides that, by virtue of the degeneracy of the genetic code, do not necessarily hybridize to the invention nucleic acids under specified hybridization conditions.
  • Preferred nucleic acids encoding the invention polypeptide are comprised of nucleotides that encode substantially the same amino acid sequence set forth in Figures 4, 8, 11 and 15.
  • nucleic acids encoding the invention polypeptide(s) hybridize under high stringency conditions to substantially the entire sequence, or substantial portions (i.e., typically at least 12 to 60 nucleotides) of the nucleic acid sequence set forth in Figures 3, 4, 8, 11 and 15, respectively.
  • the present invention provides isolated polynucleotides operatively linked to a promoter of RNA transcription, as well as other regulatory sequences.
  • operatively linked refers to the functional relationship of the polynucleotide with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
  • operative linkage of a polynucleotide to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter, and wherein the promoter directs the transcription of RNA from the polynucleotide.
  • Promoter regions include specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. Additionally, promoter regions include sequences that modulate the recognition, binding and transcription initiation activity of RNA polymerase. Such sequences may be cis acting or may be responsive to trans acting factors . Depending upon the nature of the regulation, promoters may be constitutive or regulated.
  • promoters are SP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, and the like.
  • CMV cytomegalovirus
  • MMTV mouse mammary tumor virus
  • MMLV Moloney murine leukemia virus
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vi tro or in vivo, and are commercially available from sources such as Stratagene (La Jo11a, CA) and Promega Biotech (Madison, WI) . In order to optimize expression and/or in vi tro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
  • consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
  • alternative codons encoding the same amino acid, can be substituted for coding sequences of the human netrin, human ABC3 transporter, the human ribosomal L3 subtype, or the human augmenter of liver regeneration polypeptide in order to enhance transcription (e.g., the codon preference of the host cell can be adopted, the presence of G-C rich domains can be reduced, and the like) .
  • vectors are viruses, such as baculoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • viruses such as baculoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • Polynucleotides are inserted into vector genomes using methods well known in the art.
  • insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA.
  • an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following:a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vi tro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability transcription termination and RNA processing signals
  • expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine- Dalgarno sequence and the start codon AUG (Sambrook et al . , supra . ) .
  • This invention provides a transformed host cell that recombinantly expresses the human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptides.
  • Invention host cells have been transformed with a polynucleotide encoding a human netrin, a human ABC3 transporter, a human ribosomal L3 subtype, or a human augmenter of liver regeneration polypeptide.
  • An example is a mammalian cell comprising a plasmid adapted for expression in a mammalian cell.
  • the plasmid contains a polynucleotide encoding human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide and the regulatory elements necessary for expression of the invention protein.
  • Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, plant cells, insect cells' and animal cells, especially mammalian cells. Of particular interest are E. coli , B. Subtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora , and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines.
  • Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, artificial chromosomes, and the like.
  • a large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, and the like, are known in the art.
  • host cells can be used as a source of recombinantly produced hNET, hABC3, RPL3L (formerly SEM L3) and/or hALR.
  • Nucleic acids (polynucleotides) encoding invention polypeptides may also be incorporated into the genome of recipient cells by recombination events.
  • such a sequence can be microinjected into a cell, and thereby effect homologous recombination at the site of an endogenous gene encoding hNET, hABC3, RPL3L (formerly SEM L3), and/or hALR an analog or pseudogene thereof, or a sequence with substantial identity to a hNET-, hABC3-, RPL3L (SEM L3-), or hALR- encoding gene.
  • Other recombination-based methods such as nonhomologous recombinations or deletion of endogenous gene by homologous recombination, especially in pluripotent cells, may also be used.
  • invention polypeptides and/or proteins include any natural occurring allelic variant, as well as recombinant forms thereof. Invention polypeptides can be isolated using various methods well known to a person of skill in the art.
  • the methods available for the isolation and purification of invention proteins include, precipitation, gel filtration, and chromatographic methods including molecular sieve, ion-exchange, and affinity chromatography using e.g. hNET-, hABC3-, RPL3L- (SEM L3-) , and/or hALR- specific antibodies or ligands.
  • hNET-, hABC3-, RPL3L- (SEM L3-) e.g. hNET-, hABC3-, RPL3L- (SEM L3-) , and/or hALR- specific antibodies or ligands.
  • SEM L3- SEM L3-
  • the recombinant expression vector may comprise additional sequences that encode additional amino-terminal or carboxy- terminal amino acids; these extra amino acids act as "tags" for immunoaffinity purification using immobilized antibodies or for affinity purification using immobilized ligands.
  • Peptides comprising hNET-, hABC3-, RPL3L- (SEM L3-) or hALR-specific sequences may be derived from isolated larger hNET, hABC3, RPL3L (SEM L3) , or hALR polypeptides described above, using proteolytic cleavages by e.g. proteases such as trypsin and chemical treatments such as cyanogen bromide that are well-known in the art.
  • proteases such as trypsin
  • chemical treatments such as cyanogen bromide that are well-known in the art.
  • peptides up to 60 residues in length can be routinely synthesized in milligram quantities using commercially available peptide synthesizers.
  • An example of the means for preparing the invention polypeptide(s) is to express polynucleotides encoding hNET, hABC3, RPL3L (SEM L3) , and/or hALR in a suitable host cell, such as a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte) , an insect cell (i.e., drosophila) or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods.
  • a suitable host cell such as a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte) , an insect cell (i.e., drosophila) or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods.
  • Invention polypeptides can be isolated directly from cells that have been transformed with expression vectors, described below in more detail.
  • polypeptide, biologically active fragments, and functional equivalents thereof can also be produced by chemical synthesis.
  • biologically active fragment refers to any portion of the polypeptide represented by the amino acid sequence in Figures 4, 8, 11 and 15 that can assemble into an active protein.
  • Synthetic polypeptides can be produced using Applied Biosystems, Inc. Model 43OA or 431A automatic peptide synthesizer (Foster City, CA) employing the chemistry provided by the manufacturer.
  • nucleic acids, polynucleotides, polypeptides, peptides or proteins with the following phrases: "recombinantly expressed/produced”, “isolated”, or “substantially pure”, encompasses nucleic acids, polynucleotides, polypeptides, peptides or proteins that have been produced in such form by the hand of man, and are thus separated from their native in vivo cellular environment.
  • the recombinant nucleic acids, polynucleotides, polypeptides, peptides and proteins of the invention are useful in ways that the corresponding naturally occurring molecules are not, such as identification of selective drugs or compounds.
  • cDNA sequences will be from the carboxyl end-encoding portion of the cDNA, and most preferably will include predicted transmembrane domain-encoding portions of the cDNA sequence. Transmembrane domain regions can be predicted based on hydropathy analysis of the deduced amino acid sequence using, for example, the method of Kyte and Doolittle ⁇ J. Mol . Biol . 157:105, 1982) .
  • the phrase "specifically hybridizing” encompasses the ability of a polynucleotide to recognize a sequence of nucleic acids that are complementary thereto and to form double-helical segments via hydrogen bonding between complementary base pairs.
  • Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable agent, such as a radioisotope, a fluorescent dye, and the like, to facilitate detection of the probe.
  • Invention probes are useful to detect the presence of nucleic acids encoding human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptides.
  • the probes can be used for in si tu hybridizations in order to locate biological tissues in which the invention gene is expressed.
  • synthesized oligonucleotides complementary to the nucleic acids of a polynucleotide encoding human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptides are useful as probes for detecting the invention genes, their associated mRNA, or for the isolation of related genes using homology screening of genomic or cDNA libraries, or by using amplification techniques well known to one of skill in the art.
  • antisense oligonucleotides having a sequence capable of binding specifically with any portion of an mRNA that encodes human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide so as to prevent translation of the mRNA.
  • the antisense oligonucleotide may have a sequence capable of binding specifically with any portion of the sequence of the cDNA encoding human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide.
  • compositions comprising an amount of the antisense oligonucleotide, (SAOC) , effective to reduce expression of the human netrin, the human ABC3 transporter, the human ribosomal L3 subtype, or the human augmenter of liver regeneration polypeptide by passing through a cell membrane and binding specifically with mRNA encoding the human netrin, the human ABC3 transporter, the human ribosomal L3 subtype, or the human augmenter of liver regeneration polypeptide so as to prevent its translation and an acceptable hydrophobic carrier capable of passing through a cell membrane are also provided herein.
  • SAOC antisense oligonucleotide
  • the acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type.
  • the structure may be part of a protein known to bind to a cell-type specific receptor.
  • This invention provides a means to modulate levels of expression of invention polypeptides by the use of a synthetic antisense oligonucleotide composition (SAOC) which inhibits translation of mRNA encoding these polypeptides.
  • SAOC synthetic antisense oligonucleotide composition
  • Synthetic oligonucleotides, or other antisense chemical structures designed to recognize and selectively bind to mRNA are constructed to be complementary to portions of the nucleotide sequences shown in Figures 3, 4, 8, 11 and 15, of DNA, RNA or chemically modified, artificial nucleic acids.
  • the SAOC is designed to be stable in the blood stream for administration to a subject by injection, or in laboratory cell culture conditions.
  • the SAOC is designed to be capable of passing through the cell membrane in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SAOC which render it capable of passing through cell membranes, for example, by designing small, hydrophobic SAOC chemical structures, or by virtue of specific transport systems in the cell which recognize and transport the SAOC into the cell.
  • the SAOC is designed to inactivate the target mRNA sequence by either binding to the target mRNA and inducing degradation of the mRNA by, for example, RNase I digestion, or inhibiting translation of the mRNA target by interfering with the binding of translation-regulating factors or ribosomes, or inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups which either degrade or chemically modify the target mRNA.
  • SAOCs have been shown to be capable of such properties when directed against mRNA targets (see Cohen et al . , TIPS, 10:435, 1989 and Weintraub, Sci . American, January pp.40, 1990) .
  • This invention further provides a composition containing an acceptable carrier and any of an isolated, purified human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide, an active fragment thereof, or a purified, mature protein and active fragments thereof, alone or in combination with each other.
  • acceptable carrier encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • polypeptides of the present invention can be used as the immunogen in generating such antibodies.
  • synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens.
  • natural or synthetic hNET-, hABC3-, RPL3L- (SEM L3-), and/or hALR-derived peptides are used to induce a hNET-, hABC3-, RPL3L- (SEM L3-) , and/or hALR- specific immune response
  • the peptides may be conveniently coupled to an suitable carrier such as KLH and administered in a suitable adjuvant such as Freund's.
  • selected peptides are coupled to a lysine core carrier substantially according to the methods of Tarn, Proc . Natl . Acad. Sci , USA 85:5409-5413, 1988.
  • the resulting antibodies may be modified to a monovalent form, such as, for example, Fab, Fab 2 , FAB', or FV.
  • Anti-idiotypic antibodies may also be prepared using known methods.
  • normal or mutated hNET, hABC3, RPL3L (SEM L3) , or hALR polypeptides are used to immunize mice, after which their spleens are removed, and splenocytes used to form cell hybrids with myeloma cells and obtain clones of antibody-secreted cells according to techniques that are standard in the art.
  • the resulting monoclonal antibodies are screened for specific binding to hNET, hABC3, RPL3L (SEM L3) , and/or hALR proteins or hNET-, hABC3-, RPL3L- (SEM L3-) , and/or hALR-related peptides.
  • antibodies may be used to block the function of the hNET, hABC3, RPL3L (SEM L3) , and/or hALR polypeptide, whether normal or mutant, or to perform rational drug design studies to identify and test inhibitors of the function (e.g., using an anti-idiotypic antibody approach) .
  • Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide.
  • Altered antibodies such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods described, for example, in Sambrook et al . , supra . , and Harlow and Lane, supra . Both anti-peptide and anti-fusion protein antibodies can be used, (see, for example, Bahouth et al . , Trends Pharmacol . Sci . 12:338, 1991; Ausubel et al . , supra . ) .
  • Invention antibodies can be used to isolate invention polypeptides. Additionally, the antibodies are useful for detecting the presence of the invention polypeptides, as well as analysis of polypeptide localization, composition, and structure of functional domains. Methods for detecting the presence of a human netrin, a human ABC3 transporter, a human ribosomal L3 subtype, or a human augmenter of liver regeneration polypeptide comprise contacting the cell with an antibody that specifically binds to the polypeptide, under conditions permitting binding of the antibody to the polypeptide, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of the invention polypeptide on the cell. With respect to the detection of such polypeptides, the antibodies can be used for in vi tro diagnostic or in vivo imaging methods.
  • Immunological procedures useful for in vi tro detection of the target human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide in a sample include immunoassays that employ a detectable antibody.
  • immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art.
  • An antibody can be made detectable by various means well known in the art.
  • a detectable marker can be directly or indirectly attached to the antibody.
  • Useful markers include, for example, radionuclides, enzymes, fluorogens, chromogens and chemiluminescent labels.
  • a detectable antibody can be administered to a subject and the binding of the antibody to the invention polypeptide can be detected by imaging techniques well known in the art.
  • Suitable imaging agents include, for example, gamma-emitting radionuclides such as 1:L1 In, 99m Tc, 51 Cr and the like, as well as paramagnetic metal ions, which are described in U.S. Patent No. 4,647,447.
  • the radionuclides permit the imaging of tissues by gamma scintillation photometry, positron emission tomography, single photon emission computed tomography and gamma camera whole body imaging, while paramagnetic metal ions permit visualization by magnetic resonance imaging.
  • the invention provides a transgenic non-human mammal that is capable of expressing nucleic acids encoding a human netrin, a human ABC3 transporter, a human ribosomal L3 subtype, or a human augmenter of liver regeneration polypeptide. Also provided is a transgenic non-human mammal capable of expressing nucleic acids encoding a human netrin, a human ABC3 transporter, a human ribosomal L3 subtype, or a human augmenter of liver regeneration polypeptide so mutated as to be incapable of normal activity, i.e., does not express native protein.
  • polynucleotides are DNA or cDNA having a coding sequence substantially the same as the coding sequence shown in Figures 3, 4, 8, 11 and 15.
  • non-human transgenic mammals are transgenic cows, sheep, goats, pigs, rabbits, rats and mice.
  • tissue specificity-determining elements are the metallothionein promoter and the T7 promoter.
  • Animal model systems which elucidate the physiological and behavioral roles of invention polypeptides are produced by creating transgenic animals in which the expression of the polypeptide is altered using a variety of techniques.
  • Examples of such techniques include the insertion of normal or mutant versions of nucleic acids encoding human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos to produce a transgenic animal. See, for example, Carver et al . , Bio /Techno logy 11:1263-1270, 1993; Carver et al., Cytotechnology 9:77-84, 1992; Clark et al., Bio /Technology 7:487-492, 1989; Simons et al .
  • homologous recombination of mutant or normal versions of these genes with the native gene locus in transgenic animals may be used to alter the regulation of expression or the structure of the invention polypeptides (see, Capecchi et al . , Science 244:1288, 1989; Zimmer et al . , Nature 338:150, 1989) . Homologous recombination techniques are well known in the art.
  • Homologous recombination replaces the native (endogenous) gene with a recombinant or mutated gene to produce an animal that cannot express native (endogenous) protein but can express, for example, a mutated protein which results in altered expression of the human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide.
  • microinjection adds genes to the host genome, without removing host genes.
  • Microinjection can produce a transgenic animal that is capable of expressing both endogenous and exogenous human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptides.
  • Inducible promoters can be linked to the coding region of the nucleic acids to provide a means to regulate expression of the transgene.
  • Tissue-specific regulatory elements can be linked to the coding region to permit tissue-specific expression of the transgene.
  • Transgenic animal model systems are useful for in vivo screening of compounds for identification of ligands, i.e., agonists and antagonists, which activate or inhibit polypeptide responses.
  • nucleic acids, oligonucleotides (including antisense) , vectors containing same, transformed host cells, polypeptides, as well as antibodies of the present invention can be used to screen compounds in vi tro to determine whether a compound functions as a potential agonist or antagonist to the invention protein.
  • vi tro screening assays provide information regarding the function and activity of the invention protein, which can lead to the identification and design of compounds that are capable of specific interaction with invention proteins.
  • a method for identifying compounds which bind to human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptides may be employed in a competitive binding assay.
  • Such an assay can accommodate the rapid screening of a large number of compounds to determine which compounds, if any, are capable of binding to invention polypeptides. Subsequently, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as modulators, agonists or antagonists of invention polypeptides.
  • transformed host cells that recombinantly express invention polypeptides can be contacted with a test compound, and the modulating effect (s) thereof can then be evaluated by comparing the human netrin, human ABC3 transporter, human ribosomal L3 subtype, or human augmenter of liver regeneration polypeptide-mediated response in the presence and absence of test compound, or by comparing the response of test cells or control cells (i.e., cells that do not express invention polypeptides) , to the presence of the compound.
  • a compound or a signal that "modulates the activity" of an invention polypeptide refers to a compound or a signal that alters the activity of the human netrin, the human ABC3 transporter, the human ribosomal L3 subtype, or the human augmenter of liver regeneration polypeptide so that the activity of the invention polypeptide is different in the presence of the compound or signal than in the absence of the compound or signal.
  • such compounds or signals include agonists and antagonists.
  • An agonist encompasses a compound or a signal that activates polypeptide function.
  • an antagonist includes a compound or signal that interferes with polypeptide function.
  • the effect of an antagonist is observed as a blocking of agonist-induced protein activation.
  • Antagonists include competitive and non-competitive antagonists.
  • a competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding.
  • a non-competitive antagonist or blocker inactivates the function of the polypeptide by interacting with a site other than the agonist interaction site.
  • Clones 16-166N (D16S277), 16-191N (D16S279), 16-198N (D16S280) and l6-140N (D16S276) were previously isolated from a cosmid library (Lerner et al . , Mamm. Genome 3:92-100, 1992) .
  • Cosmids CCMM65 (D16S84), c291 (D16S291) , CAJ42 (ATP6C) and cKG8 were recovered from total human cosmid libraries (made in-house or by Stratagene, La Jolla, CA) using either a cloned insert (CMM65) or sequence-specific oligonucleotides as probe.
  • CMM65 cloned insert
  • the c326 cosmid contig and clone 413C12 originated from a flow-sorted chromosome 16 library (Stallings et al . , Genomics 13 (4) : 1031-1039, 1992) .
  • the c326 contig was comprised of clones 2H2, 77E8, 325A11 and 325B10.
  • Chromosome walking experiments were done using a single set of membranes which contained the gridded PI library pools (Shepherd et al . , supra . 1994) .
  • the gridded filters were kindly provided by Dr. Mark Leppert and the Technology Access Section of the Utah Center for Human Genome Research at the University of Utah.
  • PI gridded membranes were screened using end probes derived from a set of chromosome 16 cosmids (see above) and PI clones as they were identified. Both RNA transcripts and bubble-PCR products were utilized as end probes.
  • Radiolabeled transcripts were generated using restriction enzyme digested cosmids or Pis ⁇ Alul, Haelll, Rsal, TaqI) as template for phage RNA polymerases T3, T7 and SP6.
  • the T3 and T7 promoter elements were present on the cosmid-derived templates while T7 and SP6 promoter sequences were contained on the Pl-based templates. Transcription reactions were performed as recommended by the manufacturer (Stratagene, La Jolla, CA) in the presence of [ ⁇ P 32 ]-ATP (Amersham, Arlington Heights, IL) .
  • Bubble-PCR products were synthesized from restriction enzyme digested Pis (Alul, Haelll, Rsal, TaqI) . Bubble adaptors with appropriate overhangs and phosphorylated 5 ' ends were ligated to digested PI DNA basically as described for YACs (Riley et al . , Nuc . Acids Res . 18:2887-2890, 1990) .
  • the sequence of the universal vectorette primer derived from the bubble adaptor sequence was 5 ' -GTTCGTACGAGAATCGCT-3 ' (SEQ ID NO:67), and differed from that of Riley and co-workers with 12 fewer 5' nucleotides .
  • Radiolabeled probes were pre-annealed to Cotl DNA as recommended (Life Technologies Inc., Gaithersburg, MD) and then hybridized to strips of nylon membrane to which were bound 10-20 ng each of the following DNAs: the cloned genomic template used to create the probe; one or more unrelated cloned genomic DNAs; cloned vector (no insert) ; and human genomic DNA.
  • Hybridizations were performed in CAK solution (5x SSPE, 1% SDS, 5x Denhardt's Solution, 100 mg/mL torula RNA) at 65°C overnight. Individual end probes were present at a concentration of 5xl0 5 cpm/mL. Hybridized membranes were washed to a final stringency of 0.lx SSC/0.1% SDS at 65° C. The hybridization results were visualized by autoradiography. Probes which hybridized robustly to their respective cloned template while not hybridizing to unrelated cloned DNAs, vector DNA or genomic DNA were identified and used to screen the gridded PI filters.
  • Hybridization to the arrayed PI pools was performed as described for the nylon membrane strips (above) except that multiple probes were used simultaneously. Positive clones were identified, plated at a density of 200-500 cfu per 100 mm plate (LB plus 25 mg/mL kanamycin) , lifted onto 82 mm HATF membranes (Millipore, Bedford, MA), processed for hybridization (Sambrook et al . , supra . ) and then rescreened with the complex probe mixture. A single positive clone from each pool was selected and replated onto a master plate.
  • PI DNA dot blots were prepared and each hybridized to individual radiolabeled probes. All hybridizations contained a chromosome 16pl3.3 reference probe, e.g. cAJ42, as well as a uniquely labeled PI DNA probe.
  • Ligations were performed in triplicate using 50 ng of vector DNA and 1, 3 or 6 mass equivalents of digested Pi DNA. Transformations were performed following an overnight 16°C incubation, with 1/10 and 1/2 of the transformation being plated on LB (ampicillin) plates. After overnight growth at 37°C, colonies were scraped off those plates having the highest transformation efficiency (based on a comparison to "no insert" ligation controls) and miniprepped using the alkaline lysis method. To examine the proportion of the pSPL3B containing insert, a small portion of the miniprep was digested with Hindlll, which cuts pSPL3B on each side of the multiple cloning site.
  • Cytoplasmic RNA was isolated 48 hours post-transfection.
  • the transfected COS-7 cells were removed from tissue culture dishes using 0.25% trypsin/1 mM EDTA (Life Technologies Inc., Gaithersburg, MD) . Trypsinized cells were washed in DMEM/10% FCS and resuspended in 400 ⁇ l of ice cold TKM (10 mM Tris-HCl pH 7.5, 10 mM KC1, 1 mM MgCl 2 ) supplemented with 1 ⁇ l of
  • RNAsin Promega, Madison, WI
  • Triton X-100 Triton X-100
  • the cells were incubated for 5 min. on ice.
  • the nuclei were removed by centrifugation at 1200 rpm for 5 min. at 4°C.
  • Thirty microliters of 5% SDS was added to the supernatant, with the cytoplasmic RNA being further purified by three rounds of extraction using phenol/chloroform/isoamyl alcohol (24:24:1) .
  • the cytoplasmic RNA was ethanol precipitated and resuspended in 50 ⁇ l of H 2 0.
  • Reverse transcription and PCR were performed on the cytoplasmic RNA prepared above as described (Church et al . , supra . 1994) using commercially available exon trapping oligonucleotides (Life Technologies Inc., Gaithersburg, MD) .
  • the resulting CUA-tailed products were shotgun subcloned into pAMPlO as recommended by the manufacturer (Life Technologies Inc.) . Random clones from each ligation were analyzed by colony PCR using secondary PCR primers (Life Technologies Inc.) .
  • Miniprep DNA containing the pAMPlO/exon traps was prepared from overnight cultures by alkaline lysis using the EasyPrep manifold or a QIAwell 8 system according to the manufacturers' instructions (Pharmacia, Pistcataway, NJ and Qiagen Inc., Chatsworth, CA, respectively) . DNA products containing trapped exons, based on comparison to the 177 bp "vector only" DNA product, were selected for sequencing.
  • RT-PCR reactions and/or PCR reactions were performed using different tissue-derived RNAs and/or cDNA libraries, respectively, as template with the oligonucleotide primers designed for each exon trap (above) .
  • oligonucleotides designed from the exons were then used in one or more of the following positive selection formats to screen the corresponding tissue-specific cDNA library.
  • RACE rapid amplification of cDNA ends
  • the first oligonucleotide primer was biotinylated and used for direct selection, while the second oligonucleotide was used in the repair.
  • the cloned contig was also screened using cDNA selection essentially as described (Parimoo et al . , Anal . Biochem. 228:1-17 1995), using the genomic Pi clones from this interval (Dackowski et al . , Genome Res . 6:515-524, 1996) .
  • Other coding sequence was obtained by SAmple SEquencing (SASE) .
  • hNET A random shotgun library was prepared from the 53.8B Pi clone ( Figure 18) by subcloning randomly sheared PI DNA into the pAMPlO vector (Life Technologies Inc., Gaithersburg, MD) essentially as described (Andersson et al . , (1994) Anal . Biochem . 218:300-308) . Pi DNA was randomly sheared using a nebulizer (Hudson RCI, Temecula, CA) . The library was initially screened with a 6 kb Xhol fragment, which had been shown to contain the netrin encoding exon traps ( Figure 18) .
  • the genomic sequence was edited and assembled using Sequencher (GeneCode ⁇ , Ann Arbor, MI) .
  • the coding region was predicted using the World Wide Web version of the GRAIL2 program (Uberbacher and Mural (1991) Proc . Natl . Acad. Sci . , USA 88:11261-11265; Xu et al . (1994) Genet. Eng. N. Y. 16:241-253) and a MacVector (Oxford Molecular Group, Cambell, CA) Pustell DNA/protein matrix analysis comparing the genomic sequence (translated in all reading frames) to the chicken netrins. Database searches were performed using BLASTN (Altschul et al . (1990) J. Mol . Biol . 215:403-410) and BLASTX (Altschul et al . , 1990, supra ; Gish and States (1993) Nat . Genet. 3:266-272) .
  • RT-PCR Both adult (brain, heart, kidney, leukocytes, liver, lung, a lymphoblastoid cell line, placenta, spleen, and testis) and fetal (kidney and brain) cD ⁇ A libraries were prescreened for the presence of netrin cD ⁇ As by PCR as described (Van Raay et al. , 1996, supra) . Nested RT-PCR was utilized to clone transcribed sequences from the netrin gene. Briefly, spinal cord polyA+ RNA (Clontech, Palo Alto, CA) was reverse transcribed using random primers as described (Kawasaki, 1990 In “PCR Protocols: A Guide to Methods and Applications” (M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White. Eds.), pp. 21-27, Academic Press, Inc., San Diego) .
  • Primers for PCR were designed based on the exons predicted from the analysis of the genomic sequence and used to amplify spinal cord RNA since spinal cord has been previously shown to express low levels of chicken netrin (Serafini et al . supra . ) . Nested PCR was required to detect RT-PCR products from human spinal cord RNA. Spinal cord RNA was reverse transcribed with random primers and primary PCR was performed in the presence of 2.5 M betaine (Sigma Chemical Co., St. Louis, MO) using the primers designed from the gene model (Table IV) .
  • the primary PCR reactions were then diluted 1:20 and secondary PCR was performed on 1 ⁇ L of the diluted primary reactions using nested primers (also designed from the gene model) , again in the presence of betaine.
  • nested primers also designed from the gene model
  • the inclusion of betaine at a final concentration of 2.5 M in the PCR reactions dramatically increased the purity and yield of the human netrin RT-PCR products (see, for example, International Publication No. WO 96/12041; Reeves et al . (1994) Am. J. Hum. Genet . 55:A238; Baskaran et al . (1996) Genome .Research 6:633-638) .
  • RT-PCR products were subcloned using pGEM-T (Promega, Madison, WI) as recommended by the manufacturer.
  • the resulting RT-PCR clones were sequenced with vector primers and internal primers using the ABI dye terminator chemistry (Perkin Elmer, Foster City, CA) and an ABI 377 automated sequencer (Perkin Elmer, Foster City, CA) .
  • Multiple sequence alignments were performed using ClustalW (Thompson et al . , (1994) Nucleic Acids Res . 22:4673-4680) .
  • hNET contains at least six exons.
  • the RT- PCR data indicate that the fourth predicted exon is actually split by an intron in the human netrin gene and is present as two exons .
  • Three of the RT-PCR exons were shown to be identical to the original exon traps. Aside from the extra exon, the gene model is nearly identical to the RT- PCR products.
  • the cDNA coding sequence, predicted protein product and full length sequence are shown in Figures 4A through 4C, respectively.
  • hABC3 A human lung cDNA library (LTI, Gaithersburg, MD) was screened with the GeneTrapper system (LTI, Gaithersburg, MD) using capture and repair oligonucleotides (5 '-CATTGCCCGTGCTGTCGTG-3 ' (SEQ ID NO:52) and 5 ' -CATCGCCGCCTCCTTCATG-3 ' (SEQ ID NO:53), respectively) designed from trapped exon L48757, the 5" most trapped exon with homology to murine ABCl. Direct cDNA library screening was also performed using an RT-PCR clone as probe. 5' RACE (Frohman, M.A. in Methods Enzymol . (J.N. Abelson and M.I. Simon Eds.) pp. 340-356, Academic Press, San Diego, CA 1993) was used to isolate additional 5' sequences from the ABC3 transcript.
  • capture and repair oligonucleotides 5 '-CATTGCCCGTGCTGTCGTG-3
  • RT-PCR products containing 3.3 kb of coding sequence were cloned (Table I and Figure 16) .
  • An additional RT-PCR primer was designed from a region of identity between the selected cDNA and the SASE data (Table I) .
  • a 900 bp RT-PCR clone was obtained using the latter primer in conjunction with a trapped exon derived primer. In total, 4.2 kb of coding sequence was obtained using RT-PCR.
  • clone ABCgt.l lacks 147 bp of sequence found in the RT-PCR clones and the cDNA clone ABC.5.
  • the additional 147 bp segment is likely to be the result of alternative splicing, in that it does not interrupt the open reading frame.
  • the presence of both transcript populations has been confirmed by PCR using primers flanking the alternatively spliced exon.
  • a 6.4 kb cDNA has been assembled for the hABC3 transporter.
  • the assembled cDNA contains a 5116 nucleotide long open reading frame encoding 1705 amino acids, with the predicted protein having a molecular weight of 191 kDa.
  • the proposed start methionine is 50 bp upstream of the 5' end of clone ABCgt.l. Although the sequence surrounding the start methionine matches the Kozak sequence in only 6 of 10 positions (Kozak, J " . Cell Biol . 115:887-903, 1991), the two positions which have been shown to be critical for function (an A at -3 and a G at +4) are conserved in hABC3.
  • the hABC3 cDNA contains a 792 bp 3 ' UTR with a consensus polyadenylation/cleavage site 20 bp upstream of the polyA tract.
  • a 6.8 kb transcript is detected by a 3 ' UTR cDNA probe on northern blots with highest levels of expression being observed in lung with lesser amounts in brain, heart, and pancreas. Significantly lower levels of expression were observed in placenta and skeletal muscle after longer exposure times. The ABC3 transcript was not detected in either liver or kidney.
  • RPL3L (SEM L3) : The longest cDNA is 1548 nucleotides in length ( Figure 11) . All three cDNAs have an open reading frame (ORF) of 1224 nucleotide with the longest cDNA containing a 48 nucleotide 5' untranslated region. An inframe stop codon at position 7 is followed by the Kozak initiation sequence CCACCATGT (SEQ ID NO:68) (Kozak, supra . ) . The 3' UTR for each of the three cDNAs vary in length, and lacks a consensus polyadenylation cleavage site.
  • hALR Sequences were cloned from the human ALR gene by 3' RACE using primers (e.g., external 5'- TGGCCCAGTTCATACATTTA-3 ' (SEQ ID NO:69) and internal 5'- TTACCCCTGTGAGGAGTGTG-3 ' (SEQ ID NO:70)) designed from the exon trap. A total of 468 bp have been obtained from the human ALR gene ( Figure 13) .
  • hNET hNET cDNA has at least 210 bp of 5' untranslated sequence, a 5' start methionine codon, a 3' stop codon (TGA) and is predicted to be 580 amino acids in length (Figure 4) , with the common domain structure of the netrin family being conserved ( Figure 20A) .
  • the human netrin was found to have higher homology to chicken netrin-2 than netrin-1, i.e., 56.3% versus 53.9%.
  • the region of greatest conservation includes the three EGF repeats, while the C-terminal domains are less well conserved ( Figure 20A) .
  • the EGF repeats are 78.7% and 82.2% identical between the human netrin and chicken netrin-1 and netrin-2, respectively, and 66.3% identical when compared to UNC-6.
  • the C-terminal domains of the human netrin and chicken netrin -1 and -2 are 41.9% and 42.5% indentical, respectively with the same domain of UNC-6 being only 29.4% identical to human netrin.
  • the human netrin more closely resembles the chicken netrins and UNC-6 than Drosophila NETA and NETS, since NETA contains an expansion in the C-domain while NETS contains additional sequences in the VI and V-1 domains (Harris et al . , 1996, supra ; Mitchell et al . , 1996, supra) .
  • the Structure of the Netrin Genes is conserveed Between Drosophila and Human
  • the coding regions of the two Drosophila netrin genes have been shown to be highly conserved with each being disrupted by six introns that occur in homologous sites (Harris et al . , 1996, supra) .
  • the position of five of the six Drosophila introns was found to be conserved in the human gene ( Figure 20B) .
  • the UNC-6 gene contains 12 introns in the coding region (Ishii et al . , 1992, supra) , the position of five of which correlate with the positions of the introns in the human gene.
  • the sixth Drosophila intron that does not have a counterpart in the human gene and is the only intron from Drosophila that is not conserved in the UNC-6 gene.
  • hABC3 Database searches revealed homology between ABC3 and murine ABCl and ABC2 (Luciani et al . , supra . 1994) . In addition to the murine ABCl and ABC2 proteins, ABC3 also shows homology to the putative C. elegans protein encoded by the cosmid sequence of C48B4.4 (Wilson et al . , supra . ) . Overall, ABC3 , ABCl, ABC2 and sequences encoded by C. elegans cosmid C48B4.4 have highest homology in the regions surrounding the ATP binding cassettes ( Figure 17) .
  • linker domain when one compares the sequence between the first ATP binding cassette and the second transmembrane domain, referred to as the linker domain (Luciani et al . , supra . 1994) , ABC3 shares much lower homology to these same 3 proteins listed above (amino acids 765-1044 in ABC3 in Figure 17) .
  • the linker domain of ABC3 is approximately 200 residues shorter than the linker domain present in ABCl and ABC2. Consequently, an optimum protein alignment positions a gap in the ABC3 sequence immediately C-terminal of a conserved HHl hydrophobic domain (Luciani et al . , supra . 1994) , located at position 917 through 959 in ABC3 ( Figure 17) .
  • ABC3 protein sequence revealed additional similarities to the ABC1/ABC2 subfamily. Based on PSORT analysis (Nakai et al . , supra . ) , the ABC3 protein does not appear to contain an N-terminal signal sequence and is likely to be a Type III membrane protein (Singer, Annu . Rev. Cell Biol . 6:247-296 1990) , with sequences N-terminal of the first transmembrane domain being located in the cytoplasm ( Figure 17) . Similar topography has been described for ABCl (Luciani et al . , supra . 1994) and all other ABC transported described to date (Higgins, supra . 1992) .
  • murine ABCl and ABC2 have been shown to contain a novel hydrophobic region, HHl, within the conserved linker domain.
  • HHl novel hydrophobic region
  • the HHl domain is not well conserved at the amino acid level in ABC3, an HHl domain does appear to be present within the linker region based on hydrophilicity analysis .
  • a similar HHl domain is also found in sequences encoded by cosmid C48B4.4 from C. elegans . In all these cases, the HHl domain is predicted to have a ⁇ -sheet conformation.
  • the first targeting site is the 21 amino acid N-terminal oligopeptide.
  • the serine and arginine present at positions 13 and 19 respectively, in human, bovine and murine L3 are replaced with histidines in RPL3L (SEM L3) ( Figure 12) .
  • the second potential nuclear targeting site is the bipartite motif.
  • the human, bovine and murine proteins have a KKR- (aa) 12 -KRR at position 341-358 while the SEM L3 gene has KKR- (aa) 10 -HHSRQ at position 341-358.
  • hALR hALR cDNA sequences encode a 119 amino acid protein which is 84.8% identical and 94.1% similar to the rat ALR protein (see, Figures 13 and 14) .
  • MOLECULE TYPE peptide (XI) SEQUENCE DESCRIPTION: SEQ ID NO • 1 :
  • Gly Asn lie Val Thr Pro Glu Leu Glu Val Ser Gly His Ser Ala Leu 20 25 30
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:7 :
  • MOLECULE TYPE peptide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO.17.
  • MOLECULE TYPE DNA (genomic)
  • TCCACTGTCC CAGAATGATG ATCTCAGCCC
  • CCATAGTCCC CCCAGGGTTC
  • CTGTGCATCT GTGGCTGTCA CATGCAGATG TGTGGCAAGG AGAAGGTGCC CACCAGCCAG 1260
  • TGCTTCCCCG CACCCCTGGC CCAGCCTGAT GGCAGCGGCC TTCTGGCCTT CAGCATGCAG 3720
  • CTGTGACTCG CACTGCAAAC CTGCCCGTGG CAGCTACCGC ATCAGCCTAA AGAAGTTCTG 4920
  • Trp Tyr Met Glu Ala Val Phe Pro Gly Gin Phe Gly Val Pro Gin Pro 450 455 460

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