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PubMed1990

Analysis of the human ornithine aminotransferase gene family.

Ornithine aminotransferase is a mitochondrial matrix enzyme that is deficient in patients with gyrate atrophy, an autosomal recessive disease of the eye. Southern blots of human DNA probed with a previously characterized OAT cDNA showed a complex pattern of gene fragments, suggesting a gene family. Hybridization of these blots with 5' and 3' OAT cDNA probes indicated that there are at least three to four copies of the OAT (approximately 22 kbp) and OAT-related gene sequence(s). We have isolated and partially characterized human OAT gene clones from total genomic and X-chromosome DNA libraries. Sequence analysis confirmed the following previously reported findings on the functional OAT gene: 11 exons, ten introns, an atypical TATA box (TTTAA), two CCAAT boxes, several GC-rich binding sites, 5' sequence homologous to SV40 enhancer core sequence (GTGGA/GA/GA/GG) and promoter region of three urea cycle enzymes (GTATCCTGCCCTC). In addition, we extended the OAT gene sequence in both the 5' and 3' directions and found its promoter region also contained a sequence homologous to the progesterone receptor (TGTTCA/TCC/T), several of the glucocorticoid responsive element (AGAACA), a cyclic AMP-responsive element (TGACGTCG), and recognition motifs for transcription factors AP-2, NF1 and Sp1. Partial sequence analyses of X-chromosome clones demonstrated an intron-less pseudogene with 77% identity to the functional OAT gene. These results demonstrate that the OAT gene is a gene family that contains both functional and related OAT gene sequence(s).

Amino Acid Sequence↗

PubMed1989

The type-specific epitopes of the Epstein-Barr virus nuclear antigen 2 are near the carboxy terminus of the protein.

The Epstein-Barr virus nuclear antigen 2 (EBNA 2) shows serotype variation and two serologically distinct groups of viruses have been identified. These correspond to the two hybridization groups of viruses (A and B) that are distinguished by a highly substituted nucleic acid sequence in the middle of the open reading frame of the EBNA 2 gene. An epitope survey of the EBNA 2-coding region was carried out using a new prokaryotic expression vector tailored to express DNA fragments from the M13 sequencing libraries of the B95-8 (type A) and Jijoye (type B) prototype virus strains. Short overlapping stretches of EBNA 2 sequence were expressed as fusion proteins and used in Western blotting with human sera that contained serotype-specific antibodies. The type A-specific epitope was located between residues 378 and 435 of the B95-8 EBNA 2 polypeptide and the type B-specific epitope mapped between residues 390 and 454, at the carboxy terminus of the Jijoye polypeptide chain. All of the type-specific anti-EBNA 2 sera tested reacted with fusion proteins containing one or other of these epitopes. Despite the direct correlation between the hybridization and serological phenotypes, the type-specific epitopes appear to lie in the relatively conserved carboxy-terminal region of EBNA 2. There was no indication that the residues of the non-homologous region contributed to the formation of antibody-combining sites.

Amino Acid Sequence↗

PubMed1990

Cloning and expression of a chicken c-rel cDNA: unlike p59v-rel, p68c-rel is a cytoplasmic protein in chicken embryo fibroblasts.

We isolated and sequenced a 3727 bp clone of the c-rel proto-oncogene from a chicken embryo fibroblast (CEF) cDNA library. Sequence comparison to the retroviral oncogene v-rel showed conclusively that the v-rel protein is truncated at both the amino- and carboxy-termini as compared to the c-rel protein. In vitro transcription and translation of this clone yielded a 68,000 dalton polypeptide that co-migrated on SDS polyacrylamide gels with p68c-rel from avian spleen cells. We inserted this c-rel cDNA clone into an avian retroviral vector (pJD214c-rel), and over-expressed p68c-rel in CEF. Over-expression of p68c-rel did not induce morphological transformation of these cells. Unlike p59v-rel, which is a nuclear protein in CEF, indirect immunofluorescence showed that p68c-rel in JD214c-rel infected CEF is located exclusively in the cytoplasm of these cells, even though the sequence of p68c-rel showed that it contains a nuclear localizing sequence identical to the one previously identified in p59v-rel. Furthermore, the c-rel protein does contain a nuclear localizing sequence which can function in CEF since replacement of the v-rel nuclear localizing sequence with the homologous domain from c-rel resulted in a hybrid rel protein that was located in the nucleus in CEF. Mutant c-rel proteins, deleted of the carboxy-terminal sequences not present in p59v-rel, localized to the nucleus in CEF. Our results show that the carboxy-terminus of p68c-rel inhibits nuclear localization of the protein, and suggest that subcellular location may be a form of regulation of the activity of p68c-rel.

Amino Acid Sequence↗

PubMed1986

Rat liver UDP-glucuronosyltransferase. cDNA sequence and expression of a form glucuronidating 3-hydroxyandrogens.

A cDNA clone, pUDPGTr-4, encoding a form of rat UDP-glucuronosyltransferase has been isolated from a SV40 expression library. Sequence analysis revealed that the cDNA is 1970 base pairs in length and encodes a protein of 530 amino acids, which has amino- and carboxyl-terminal sequences characteristic of signal peptide and transmembrane segments, respectively. There is one potential asparagine-linked glycosylation site. Transfection of UDPGTr-4 cDNA into COS cells resulted in the glucuronidation of etiocholanolone, androsterone, and lithocholic acid in a transient expression assay. Several other common substrates of UDP-glucuronosyltransferase were not conjugated by the UDPGTr-4 enzyme. UDPGTr-4 cDNA is identical in sequence over a common 1.7 kilobase-region of overlap to UDPGTr-1, a cDNA previously isolated in this laboratory (Mackenzie, P. I., Gonzalez, F. J., and Owens, I. S. (1984) J. Biol. Chem. 259, 12153-12160). UDPGTr-4 cDNA, however, contains a shorter 3'-untranslated region. Northern analysis showed that the poly(A) RNA counterparts of UDPGTr-4 and UDPGTr-1 cDNAs are approximately 2.3 and 3.0 kilobases in length, respectively. The steady-state level of UDPGTr-4 poly(A) RNA in the liver is 20-fold higher than that of UDPGTr-1 poly(A) RNA. These data indicate that the UDPGTr-4 enzyme is a 3-hydroxyandrogen UDP-glucuronosyltransferase which is encoded by two distinct species of mRNA transcribed from the same gene.

Amino Acid Sequence↗

PubMed1999

A shotgun optical map of the entire Plasmodium falciparum genome.

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7-2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).

Animals↗

PubMed1996

The BRCA1 and 1A1.3B promoters are parallel elements of a genomic duplication at 17q21.

The results of experiments aimed at detecting polymorphisms and mutations in the BRCA1 promoter region as well as comparisons of two published DNA sequences indicated that two similar but distinct copies of this region exist in the human genome. PCR primers specific for amplification of each of the related sequences were developed and new genomic clones corresponding to each of the two promoter regions were isolated from rearrangement-resistant libraries. Sequence analysis of the clones and specific PCR products reveals two similar genomic arrangements of head-to-head genes. The BRCA1 gene is closely apposed to a gene structure that is similar but not identical to 1A1.3B, and the 1A1.3B gene is apposed to a gene structure that has strong similarity to BRCA1 but also significant differences. STS analysis of YAC and P1 clones located in the vicinity of BRCA1 indicates that these similar promoter regions are elements of a direct duplication. New hypotheses for genetic mechanisms that may be involved in breast and ovarian cancer etiology are raised by the identification of this duplicated genetic structure on chromosome 17q.

BRCA1 Protein↗

PubMed1995

Sequence of the dog immunoglobulin alpha and epsilon constant region genes.

The immunoglobulin alpha (IGHAC) and epsilon (IGHEC) germline constant region genes were isolated from a dog liver genomic DNA library. Sequence analysis indicates that the dog IGHEC gene is encoded by four exons spread out over 1.7 kilobases (kb). The IGHAC sequence encompasses 1.5 kb and includes all three constant region coding exons. The complete exon/intron sequence of these genes is described.

Amino Acid Sequence↗

PubMed1994

Cloning and sequencing of a cDNA encoding the murine vitamin K-dependent protein S.

A cDNA clone encoding the vitamin K-dependent protein S was isolated from a murine liver cDNA library. Sequencing revealed an open reading frame encoding a 634 amino acid mature protein. Comparison to human and bovine sequences showed a high degree of similarity and the conservation of key residues between the species.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N.

Carboxypeptidase M, a widely distributed membrane-bound carboxypeptidase that can regulate peptide hormone activity, was purified to homogeneity from human placenta (Skidgel, R. A., Davis, R. M., and Tan, F. (1989) J. Biol. Chem. 264, 2236-2241). The NH2-terminal 31 amino acids were sequenced, and two complementary oligonucleotide probes were synthesized and used to isolate a carboxypeptidase M clone from a human placental cDNA library. Sequencing of the cDNA insert (2009 base pairs) revealed an open reading frame of 1317 base pairs coding for a protein of 439 residues. The NH2-terminal protein sequence matched the deduced amino acid sequence starting with residue 14. Hydropathic analysis revealed hydrophobic regions at the NH2 and COOH termini. The NH2-terminal 13 amino acids probably represent part of the signal peptide, and the COOH-terminal hydrophobic region may act either as a transmembrane anchor or as a signal for attachment to a phosphatidylinositol glycan moiety. The carboxypeptidase M sequence contains six potential Asn-linked glycosylation sites, consistent with its glycoprotein nature. The sequence of carboxypeptidase M was 41% identical with that of the active subunit of human plasma carboxypeptidase N, 41% identical with bovine carboxypeptidase H (carboxypeptidase E, enkephalin convertase), and 15% with either bovine pancreatic carboxypeptidase A or B. Many of the active site residues identified in carboxypeptidases A and B, including all of the zinc-binding residues (2 histidines and a glutamic acid), are conserved in carboxypeptidase M. These data indicate that all of the metallocarboxypeptidases are related, but the nondigestive carboxypeptidases with more specialized functions, present in cell membranes, blood plasma, or secretory granules (i.e., carboxypeptidase M, carboxypeptidase N and carboxypeptidase H), are more closely related to each other (41-49% identity) than they are to carboxypeptidase A or B (15-20% identity).

Amino Acid Sequence↗

PubMed1992

cDNA cloning and molecular characterization of MSE55, a novel human serum constituent protein that displays bone marrow stromal/endothelial cell-specific expression.

Hemonectin is a lineage-specific cytoadhesive protein that may be involved in the developmentally regulated adhesion of granulocytic cells to bone marrow stroma. Immunoblot analysis using an anti-hemonectin antibody recognizes two distinct immunoreactive species in endothelial cell lysates (approximately M(r) 65,000) and human serum (approximately M(r) 55,000). Initial characterization of the 55-kDa protein has now been completed by isolating the cDNA from a human endothelial cell expression library. Sequence analysis of overlapping clones identifies a composite sequence spanning 2030 nucleotides with an open reading frame of 1173 base pairs. No significant sequence similarity was observed on analysis of current GenBank databases. The open reading frame was expressed as a recombinant protein in Escherichia coli and used as an immunogen for the production of a specific polyclonal antibody. Immunoblotting with this antibody identifies a single immunoreactive species of apparent M(r) 55,000 in HUVEC lysates and human serum, confirming that a secreted form normally circulates as a serum constituent protein. This antibody fails to recognize purified hemonectin, suggesting that the M(r) 55,000 protein is not hemonectin. Cross-species Southern blot analysis reveals persistent hybridizing fragments in all species tested, suggestive of a developmentally conserved function. Northern blot analysis demonstrates expression limited to endothelial and bone marrow stromal cells, but not poly(A) RNA from monkey liver, spleen, brain, lung, and kidney. On this basis, we have designated this novel protein MSE55, for marrow stromal/endothelial cell protein with a molecular mass of 55,000 daltons. Its tissue-specific expression may suggest a functional role in hematopoiesis.

Adaptor Proteins, Signal Transducing↗

PubMed1996

BETA3, a novel helix-loop-helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes.

Using degenerate PCR cloning we have identified a novel basic helix-loop-helix (bHLH) transcription factor, BETA3, from a hamster insulin tumor (HIT) cell cDNA library. Sequence analysis revealed that this factor belongs to the class B bHLH family and has the highest degree of homology with another bHLH transcription factor recently isolated in our laboratory, BETA2 (neuroD) (J. E. Lee, S. M. Hollenberg, L. Snider, D. L. Turner, N. Lipnick, and H. Weintraub, Science 268:836-844, 1995; F. J. Naya, C. M. M. Stellrecht, and M.-J. Tsai, Genes Dev. 8:1009-1019, 1995). BETA2 is a brain- and pancreatic-islet-specific bHLH transcription factor and is largely responsible for the tissue-specific expression of the insulin gene. BETA3 was found to be tissue restricted, with the highest levels of expression in HIT, lung, kidney, and brain cells. Surprisingly, despite the homology between BETA2 and BETA3 and its intact basic region, BETA3 is unable to bind the insulin E box in bandshift analysis as a homodimer or as a heterodimer with the class A bHLH factors E12, E47, or BETA1. Instead, BETA3 inhibited both the E47 homodimer and the E47/BETA2 heterodimer binding to the insulin E box. In addition, BETA3 greatly repressed the BETA2/E47 induction of the insulin enhancer in HIT cells as well as the MyoD/E47 induction of a muscle-specific E box in the myoblast cell line C2C12. In contrast, expression of BETA3 had no significant effect on the GAL4-VP16 transcriptional activity. Immunoprecipitation analysis demonstrates that the mechanism of repression is via direct protein-protein interaction, presumably by heterodimerization between BETA3 and class A bHLH factors.

Amino Acid Sequence↗

PubMed1994

Multiple TL-like loci in the grc-G/C region of the rat.

The grc-G/C region of the rat is homologous to the Q/TL region of the mouse, and deletions in this region are associated with fetal mortality, developmental defects, and decreased resistance to cancer. Several cosmids spanning approximately 45 kilobases of this region were analyzed for their class I loci, using a mouse general class I probe (pAG64c), grc-specific probes (pGRC1.4, pGRC1.7), and four probes derived from the TL-like locus RT1.N1. The results showed that TL-like genes other than RT1.N1 exist in the rat: a duplicated gene, RT1.N2, was identified, sequenced, and shown to be 99.3% similar to RT1.N1; and a third TL-like gene, RT1.N3, was isolated from a cDNA library, sequenced, and shown to be 92.8% similar to RT1.N1. These observations suggest that the rat TL-like loci are duplicated and that there is more than one cluster of these duplicated genes. The TL-like genes are transcribed predominantly in the thymus, except in grc- strains, and their level of transcription increases during fetal life and reaches its maximum at birth. Finally, a cosmid that appears to identify the end of the deletion in grc- strains was identified.

Amino Acid Sequence↗

PubMed1995

Structure and chromosomal localization of the human anti-müllerian hormone type II receptor gene.

Using the rat anti-müllerian hormone type II receptor (AMHRII) cDNA as a probe, two overlapping lambda phage clones containing the AMHRII gene were isolated from a human genomic library. Sequence analysis of the exons was performed and the exon/intron boundaries were determined. The coding region was found to consist of 11 exons, divided over 8 kb. The genomic structure resembles that of the related activin type II receptor gene. The AMHRII gene was mapped to human chromosome 12q12-q13. The results reported are essential for identification of AMHRII gene alterations in patients with persistent müllerian duct syndrome.

Amino Acid Sequence↗

PubMed1987

Structure of the rat platelet factor 4 gene: a marker for megakaryocyte differentiation.

A rat platelet factor 4 (PF4) cDNA has been isolated by immunoscreening a g lambda 11 rat megakaryocyte cDNA expression library. Sequence analysis of the rat PF4 cDNA revealed that this megakaryocyte protein is composed of a leader sequence of 29 amino acid residues and a mature protein sequence of 76 amino acid residues. The structure of rat PF4 derived from its cDNA shows a marked homology with the amino acid sequence of human PF4 obtained by classical protein chemistry techniques. This observation is particularly striking with regard to the carboxy-terminal region of rat and human PF4, where 28 of the last 31 C-terminal residues are identical. The rat PF4 gene was obtained from a rat genomic library by using rat PF4 cDNA as a hybridization probe. Sequence analysis showed that the gene is constructed of three exons and two short introns. The transcriptional start site is located 73 base pairs upstream of the translational start codon as judged by S1 nuclease mapping and primer extension. The 5' noncoding region of the gene also exhibited a sequence homologous to the TATA box at -31, as well as a series of direct and inverted repeat sequences and a cluster of 26 T residues at -155 to -218. This latter domain may be involved in regulating PF4 gene expression during megakaryocytopoiesis.

Animals↗

PubMed1998

Prediction of local structure in proteins using a library of sequence-structure motifs.

We describe a new method for local protein structure prediction based on a library of short sequence pattern that correlate strongly with protein three-dimensional structural elements. The library was generated using an automated method for finding correlations between protein sequence and local structure, and contains most previously described local sequence-structure correlations as well as new relationships, including a diverging type-II beta-turn, a frayed helix, and a proline-terminated helix. The query sequence is scanned for segments 7 to 19 residues in length that strongly match one of the 82 patterns in the library. Matching segments are assigned the three-dimensional structure characteristic of the corresponding sequence pattern, and backbone torsion angles for the entire query sequence are then predicted by piecing together mutually compatible segment predictions. In predictions of local structure in a test set of 55 proteins, about 50% of all residues, and 76% of residues covered by high-confidence predictions, were found in eight-residue segments within 1.4 A of their true structures. The predictions are complementary to traditional secondary structure predictions because they are considerably more specific in turn regions, and may contribute to ab initio tertiary structure prediction and fold recognition.

Algorithms↗

PubMed1998

Cloning of a beta1 tubulin cDNA from an insect endocrine gland: developmental and hormone-induced changes in mRNA expression.

A rapid increase in ecdysteroid hormone synthesis results when the insect prothoracic gland is stimulated with prothoracicotropic hormone (PTTH), a brain neuropeptide hormone. PTTH also stimulates the specific synthesis of several proteins, one of which is a beta tubulin. To further understand the possible roles of beta tubulin in the prothoracic gland, beta tubulin cDNA clones were isolated from a tobacco hornworm (Manduca sexta) gland cDNA library. Sequence analysis indicated that these clones were assignable to the beta1 tubulin isoform. Gland beta1 tubulin mRNA levels during the last larval instar and early pupal-adult development exhibited peaks that coincided with peaks in ecdysteroid synthesis. Manipulations of the glands hormonal milieu showed that beta1 tubulin mRNA levels respond to 20 hydroxyecdysone and PTTH. The data also support our earlier proposal that the prothoracic gland beta1 tubulin gene is ubiquitously expressed but exhibits tissue- and developmental-specific regulation of transcription and translation.

Amino Acid Sequence↗

PubMed1995

An evaluation of a multicenter study on HLA-DPB1 typing using solid-phase Taq-cycle sequencing chemistry.

In HLA Class II genes, polymorphism is mainly located in the second exon. Most DNA based typing methods are confined to the identification of specific sequence motifs in the second exon. In contrast, Sequencing Based Typing (SBT) elucidates the entire exon 2 sequence for typing. Comparison of the obtained exon 2 sequence with an allele sequence library results in allele assignment. We tested the applicability of SBT using a protocol for amplification followed by solid phase Taq-cycle sequencing for HLA-DPB1 typing. A panel of 32 samples were typed by SBT at five test sites which are participating in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. In this multicenter study we focused on the reliability of analyzing heterozygous sequences for HLA typing. A multi-sequence analysis approach, Polall, was developed to evaluate sequences obtained. The assignment of homozygosity and heterozygosity was validated by cluster analysis of chromatographic data of all sequences. Sequence characteristics were examined and considered for appropriate assignment. Differences in sequence characteristics that occurred between the test sites are considered in detail. The evaluation of data of 5 test sites reveals that Taq-cycle sequencing can reliably be performed for HLA-DPB1 SBT.

DNA-Directed DNA Polymerase↗

PubMed1992

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.

Amino Acid Sequence↗