Exon-intron organization of the human gp130 gene

The exon-intron organization and sequences of the exon-intron boundaries of the human gp130 transmembrane receptor gene have been determined using genomic DNAs as samples. The gp130 gene comprises 17 exons and 16 introns. The positions of the exon-intron boundaries show good correlation to the functional/homology regions of gp130. Exons 3-17 code for the gp130 protein, and each subdomain of the receptor is encoded by a set of exons. The coding potential of exons and the intron phasing of the human gp130 gene conform to the patterns observed previously for other cytokine receptor genes. This supports the notions that the gp130 gene evolved from the same ancestral gene that gave rise to other members of the cytokine receptor family.     

Cloning and characterization of the CDZFP gene which encodes a putative zinc finger protein.

We report here the cloning and characterization of a novel human cytoplasm-distribution zinc finger protein (CDZFP) gene, isolated from human ovary cDNA library, and mapped to 4p12 by searching the UCSC genomic database. The CDZFP cDNA is 1793 base pairs in length and contains an open reading frame (ORF) encoding 236 amino acids. The CDZFP gene consists of 7 exons and encodes a putative zinc finger protein with a transmembrane region and two zinc finger motifs. Subcellular localization demonstrated that CDZFP protein was located in the cytoplasm when overexpressed in Hela cells and northern blot analysis revealed that CDZFP was ubiquitously expressed in 16 human tissues.     

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct patterns of expression of the RB gene family in mouse and human retina

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.     

Complete Structure of the Human Gc Gene: Differences and Similarities between Members of the Albumin Gene Family

The sequence of the human Gc gene, including 4228 base pairs of the 5′-flanking region and 8514 base pairs of the 3′ flanking region (55,136 in total), was determined from five overlapping λ phage clones. The sequence spans 42,394 base pairs from the cap site to the polyadenylation site, and it reveals that the gene is composed of 13 exons, which are symmetrically placed within the three domains of the Gc protein. The first exon is partially untranslated, as is exon 12, which contains the termination codon TAG. Exon 13 is entirely untranslated, but contains the polyadenylation signal AATAAA. Ten central introns split the coding sequence between codon positions 2 and 3 and between codon positions 3 and 1 in an alternating pattern, exactly as has been observed in the structure of the albumin and α-fetoprotein genes. The Gc gene has several distinctive features which set it apart from the other members of the family. First, the gene is smaller by two exons, which results in a protein some 130 amino acids shorter than albumin or AFP. This decrease in size may result from the loss of two internal exons during the evolutionary history of the Gc gene. Second, exons 6, 8, 9, and 11 are smaller than their counterparts in albumin or AFP by a total of 8 codons (1, 4, 1, and 2, respectively). Although the mRNA and protein expressed from the Gc gene are significantly smaller, the gene itself is about 2.5 times larger than the other genes of the family. There are 13 interspersed DNA repeats within the human Gc gene which are absent from the same positions in the albumin or AFP genes, and hence must have been inserted after the triplication event(s) that gave rise to the gene family. Despite the differences, the Gc gene is nonetheless recognizable as a member of the albumin family.     

Characterization of the human platelet glycoprotein IIIa gene. Comparison with the fibronectin receptor beta-subunit gene

This study was designed to determine the structure of the gene for glycoprotein (GP) GPIIIa, the beta-subunit of the platelet membrane GPIIb-IIIa complex. The complexity of the gene was determined after Southern analysis of human chromosomal DNA. Overlapping genomic clones were isolated from cosmid and phage lambda libraries that contained the entire coding unit of the human gene for the mature GPIIIa protein. The genomic clones spanned approximately 60 kilobase pairs of human DNA sequence. The exon containing segments of the clones was mapped and the exons, including the exonintron junctions, were sequenced. The GPIIIa protein is divided into 14 exons ranging in size from 87 to 430 nucleotides separated by introns, which were 0.3 to 9 kilobase pairs in size. The 3' exon was larger than 1700 nucleotides and contained the 3'-untranslated region. Several structural domains of the GPIIIa protein were contained within individual exons. These included (i) the transmembrane spanning segment, (ii) the cytoplasmic region containing the potential phosphorylation sites, and (iii) the six domains in the NH2-terminal half of GPIIIa that are highly conserved between two other integrin beta-subunits. In contrast, other domains such as the four cysteine-rich repeats were interrupted by introns. Genomic clones for the beta-subunit of the fibronectin receptor (beta 1) were also isolated, partially mapped, and sequenced. Of the eight splice sites identified in beta 1, six occurred at the same amino acid residue in GPIIIa. These results provide genetic evidence that GPIIIa and beta 1 have a common evolutionary origin within the integrin family.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.     

Genomic organization and localization of the human CRMP-1 gene.

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein considered to be involved in the collapsin-induced growth cone collapse during neural development. CRMP-1 belongs to the Unc-33 gene family. Here we report the genomic structure and the localization of the human CRMP-1 gene to chromosome 4p16.1. Sequence analysis revealed that the human CRMP-1 gene consists of 14 exons. We have also established sequencing assays for all its coding exons. This should permit the rapid screening for mutations to assess CRMP-1 role in genetic disorders mapped in the 4p16.1 region.     

Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen.

Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.     

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.     

Proteasomal degradation of retinoblastoma-related p130 during adipocyte differentiation.

Within 24 h of hormonally stimulated 3T3-L1 adipocyte differentiation, there are dramatic changes in the protein levels of p130 and p107, two members of the retinoblastoma tumor suppressor gene family. Designated the "p103:p107" switch, this alteration is characterized by a rapid and transient drop in p130 protein levels accompanied by a transient increase in both p107 mRNA and protein levels. Using protease inhibitors, the specific proteolytic pathway involved in degradation of p130 was examined. Treatment of cells with N-acetyl-leu-leu-norleucinal, an inhibitor that blocks proteolytic activity of type I calpain and the 26S proteasome, resulted in a complete block in the degradation of p130 protein, as well as adipocyte differentiation, suggesting that one of these pathways is involved in regulating p130 protein levels. Similar analysis with lactacystin, a specific inhibitor of the 26S proteasome, also resulted in a complete block in both differentiation and p130 degradation. Furthermore, both inhibitors blocked the increase in p107 protein levels normally observed on Day 1, suggesting that the p130:p107 switch is required for adipocyte differentiation and one of the early molecular events involved in activating the p130:p107 switch is the specific degradation of p130 by the 26S proteasome.     

Structure of the gene for human von Willebrand factor

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.     

Structure of the human lck gene: differences in genomic organisation within src-related genes affect only N-terminal exons

Although cDNA sequences coding for several Rous sarcoma virus Src-related protein tyrosine kinases (PTKs) have been reported for several years, knowledge of the structure and organisation of genes of the src family is still limited. In this work, a detailed structure and organisation of the human lck gene is reported. A 17-kb genomic clone encoding human p56 Lck, a lymphocyte-specific PTK of the Src-related subfamily, has been isolated. The human lck gene is organized in 13 exons, one more than in the human cellular (c)-src gene. The twelve coding exons are located in this clone, whereas the putative 5'-noncoding exon is probably located very far upstream from the second exon. Splicing sites for exons 4 to 12, which encode both conserved phospholipase-C-like and catalytic domains of the Src-like PTKs, arise exactly at the same position for the human lck, human c-src and c-fgr genes. The only differences concern the splice sites of exons 1' and 2, which encode the unique N-terminal domain of human Lck. These results give further evidence that the different PTKs of the Src-like family have probably evolved through the mechanism of exon shuffling.