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PubMed1982

Structural relationship between a normal chicken DNA locus and the transforming gene of the avian acute leukemia virus MC29.

We screened a recombinant chicken DNA/lambda phage library for sequences homologous to the transformation-specific sequences of the avian acute leukemia virus MC29 by hybridization with molecularly cloned MC29 proviral DNA. Three cellular DNA clones were found and compared with each other and with the viral genome by physical mapping with restriction endonucleases and by heteroduplex analysis. These experiments indicated that the three cellular clones overlap and represent a single cellular locus. The RNA genome of MC29 and normal cell DNA share a homologous region of 1.6 kilobases which is interrupted in the cellular DNA by 1.0 kilobase of sequences not present in the viral genome. Hybridization of the cloned cellular DNA to viral RNA and analysis of the protected viral RNA by fingerprinting techniques indicated that there is extensive sequence homology between the helper virus-unrelated mcv sequences of the viral RNA and the cellular DNA, with only minor base differences. The cellular mcv locus, however, lacks all helper virus-related sequences of MC29, including those of the partial viral gag gene which, together with mcv, encodes the probable transforming protein of MC29. We conclude that although the mcv locus of the normal cell does not represent a complete structural homolog to the onc gene of MC29, it is probably the precursor to the onc-specific sequence in the virus.

Alpharetrovirus↗
PubMed1995

Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.

Previously, we demonstrated induction of a unique macrophage prothrombinase during infection of BALB/cJ mice by mouse hepatitis virus strain 3 (MHV-3). By immunologic screening, a clone representing this prothrombinase was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp but, when infected with MHV-3, synthesized musfiblp-specific mRNA. musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrated when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.

Amino Acid Sequence↗
PubMed1997

Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain.

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response of P. includens toward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed in P. includens hemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0- and 1.5-kb cDNA clones (MdPi455 and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5' ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense against M. demolitor and other parasites.

Amino Acid Sequence↗
PubMed1998

Binding of murine leukemia virus Gag polyproteins to KIF4, a microtubule-based motor protein.

A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.

Animals↗
PubMed1985

Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.

Amino Acid Sequence↗
PubMed1987

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

Animals↗
PubMed2003

A first-generation map of the turkey genome.

A primary linkage map of the domestic turkey (Meleagris gallopavo) was developed by segregation analysis of genetic markers within a backcross family. This reference family includes 84 offspring from one F1 sire mated to two dams. Genomic DNA was digested using one of five restriction enzymes, and restriction fragment length polymorphisms were detected on Southern blots using probes prepared from 135 random clones isolated from a whole-embryo cDNA library. DNA sequence was subsequently determined for 114 of these cDNA clones. Sequence comparisons were done using BLAST searches of the GenBank database, and redundant sequences were eliminated. High similarity was found between 23% of the turkey sequences and mRNA sequences reported for the chicken. The current map, based on expressed genes, includes 138 loci, encompassing 113 loci arranged into 22 linkage groups and an additional 25 loci that remain unlinked. The average distance between linked markers is 6 cM and the longest linkage group (17 loci) measures 131 cM. The total map distance contained within linkage groups is 651 cM. The present map provides an important framework for future genome mapping in the turkey.

Animals↗
PubMed1998

Isolation of rat fibrillin-1 cDNA and its relevance in metanephric development.

The role of fibrillin-1 in metanephrogenesis was investigated. Fibrillin-1 cDNA was isolated from the rat kidney cDNA library and sequenced, and its spatiotemporal expression was studied. It had approximately 88% homology with human fibrillin-1 and had Ca2+ binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and an RGD binding site. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-1 expression was developmentally regulated. In situ hybridization and immunofluorescence studies indicated that at day 15 of gestation, fibrillin-1 is expressed in the metanephric mesenchyme. At day 18, its expression was confined to nascent blood vessels and glomeruli, and it increased in the newborn and neonatal kidneys. Immunoprecipitation revealed an approximately 300-kDa band by SDS-PAGE. Treatment with fibrillin-1 antisense oligodeoxynucleotide induced marked dysmorphogenesis of the embryonic metanephroi. Concomitantly, the fibrillin-1 mRNA, antibody reactivity in the metanephroi, and fibrillin-1-specific radioincorporation were reduced. These data indicate that, like alphavbeta3 integrin, a known morphogen and a putative receptor of fibrillin-1, the fibrillin-1 modulates events related to early organogenesis and possibly also the vascularization of the rat kidney.

Amino Acid Sequence↗
PubMed2001

Genetic mapping and functional studies of a natural inhibitor of the insulin receptor tyrosine kinase: the mouse ortholog of human alpha2-HS glycoprotein.

Fetuin/alpha2-HS glycoprotein (alpha2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron-exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n*(A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse alpha2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse alpha2-HSG inhibits insulin-stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse alpha2-HSG (25 microg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene.

Amino Acid Sequence↗
PubMed2000

Genomic structure and chromosome location of the gene encoding mouse CD59.

The gene encoding the mouse analogue of the human complement regulator CD59 was cloned using a combination of long range PCR and genomic library screening. Sequence obtained showed that its genomic structure closely resembled that of the human CD59 gene, comprising 4 exons, each separated by a long intron region. The sizes of introns and exons were comparable to those of the human gene with the exception of the third intron which is 2.5 kb in the mouse compared to 7 kb in the human gene. All exon/intron boundaries conformed to the GT-AG rules for splicing. Radiation hybrid mapping localised mouse Cd59 between D2Mit333 and D2Mit127 on chromosome 2, a region homologous with human chromosome 11p13 where the human CD59 gene is localised. These data have permitted the construction of a gene targeting vector for the generation of transgenic mice deficient in CD59.

Animals↗
PubMed2000

VI. Genome structure and cognitive map of Williams syndrome.

Williams syndrome (WMS) is a most compelling model of human cognition, of human genome organization, and of evolution. Due to a deletion in chromosome band 7q11.23, subjects have cardiovascular, connective tissue, and neurodevelopmental deficits. Given the striking peaks and valleys in neurocognition including deficits in visual-spatial and global processing, preserved language and face processing, hypersociability, and heightened affect, the goal of this work has been to identify the genes that are responsible, the cause of the deletion, and its origin in primate evolution. To do this, we have generated an integrated physical, genetic, and transcriptional map of the WMS and flanking regions using multicolor metaphase and interphase fluorescence in situ hybridization (FISH) of bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), BAC end sequencing, PCR gene marker and microsatellite, large-scale sequencing, cDNA library, and database analyses. The results indicate the genomic organization of the WMS region as two nested duplicated regions flanking a largely single-copy region. There are at least two common deletion breakpoints, one in the centromeric and at least two in the telomeric repeated regions. Clones anchoring the unique to the repeated regions are defined along with three new pseudogene families. Primate studies indicate an evolutionary hot spot for chromosomal inversion in the WMS region. A cognitive phenotypic map of WMS is presented, which combines previous data with five further WMS subjects and three atypical WMS subjects with deletions; two larger (deleted for D7S489L) and one smaller, deleted for genes telomeric to FZD9, through LIMK1, but not WSCR1 or telomeric. The results establish regions and consequent gene candidates for WMS features including mental retardation, hypersociability, and facial features. The approach provides the basis for defining pathways linking genetic underpinnings with the neuroanatomical, functional, and behavioral consequences that result in human cognition.

Adolescent↗
PubMed1985

Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family.

A near full-length cDNA encoding the human vitamin D-binding protein (hDBP) was isolated from a human liver mRNA expression library. Complete sequence analysis of this clone predicts the full-length amino acid sequence of the pre-hDBP. Comparison of the sequence of the hDBP mRNA and protein to existing protein and nucleic acid data banks demonstrates a strong and highly characteristic homology of the hDBP with human albumin (hALB) and human alpha-fetoprotein (hAFP). Based upon this structural comparison, we establish that DBP is a member of the ALB and AFP gene family.

Amino Acid Sequence↗
PubMed2006

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Animals↗
PubMed2003

Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis.

Adult Leydig cell steroidogenesis is dependent on LH but fetal Leydig cells can function independently of gonadotropin stimulation. To identify factors that may be involved in regulation of fetal Leydig cells expressed sequence tag libraries from fetal and adult testes were compared, and fetal-specific genes identified. The ACTH receptor [melanocortin type 2 receptor (Mc2r)] was identified within this fetal-specific group. Subsequent real-time PCR studies confirmed that Mc2r was expressed in the fetal testis at 100-fold higher levels than in the adult testis. Incubation of fetal or neonatal testes with ACTH in vitro stimulated testosterone production more than 10-fold, although ACTH had no effect on testes from animals aged 20 d or older. The steroidogenic response of fetal and neonatal testes to a maximally stimulating dose of human chorionic gonadotropin was similar to the response shown to ACTH. The ED(50) for ACTH, measured in isolated fetal and neonatal testicular cells, was 5 x 10(-10) M and the lowest dose of ACTH eliciting a response was 2 x 10(-11) M. Circulating ACTH levels in fetal mice were around 8 x 10(-11) M. Neither alpha-MSH nor gamma-MSH had any effect on androgen production in vitro at any age. Fetal testosterone levels were normal in mice that lack circulating ACTH (proopiomelanocortin-null) indicating that ACTH is not essential for fetal Leydig cell function. Results show that both LH and ACTH can regulate testicular steroidogenesis during fetal development in the mouse and suggest that fetal Leydig cells, but not adult Leydig cells, are sensitive to ACTH stimulation.

Adrenocorticotropic Hormone↗
PubMed1993

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor.

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Amino Acid Sequence↗
PubMed1990

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin.

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.

Amino Acid Sequence↗
PubMed1993

Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level.

The insulin-dependent tyrosine kinase activity (TKA) of the insulin receptor (IR) plays an essential role in insulin signaling. Thus, dysregulation of IR-TKA might be an important element in the states of insulin resistance. A phosphorylated rat hepatic glycoprotein (pp63) acting as an inhibitor of IR-TK has been described. In search of the human homolog of pp63, we isolated a cDNA clone from a human liver lambda gt11 cDNA library. DNA sequence analysis reveals identity with the mRNA product of a human gene AHSG encoding a serum protein, alpha 2-Heremans Scmid-glycoprotein (alpha 2HSG), with heretofore unknown physiological function. Northern blot analysis demonstrates a 1.8-kilobase mRNA in human liver and HepG2 hepatoma cells. alpha 2HSG, purified from human serum, specifically inhibits insulin-stimulated IR autophosphorylation in vitro and in vivo as well as exogenous substrate tyrosine phosphorylation. alpha 2HSG also inhibits both insulin-induced tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 subunit of phosphatidylinositol-3 kinase in H-35 hepatoma cells. alpha 2HSG inhibits insulin-dependent mitogenesis, but does not affect insulin-stimulated induction of the metabolic enzyme tyrosine aminotransferase. alpha 2HSG does not compete with insulin for binding to IR. Finally, the action of alpha 2HSG is specific toward the IR-TK; its effect does not extend to insulin-like growth factor-I-stimulated TKA. Our results allow us to assign a biochemical function for human alpha 2HSG, namely regulation of insulin action at the IR-TK level.

Amino Acid Sequence↗