Structural and Functional Analyses of the Hfb1, Hmob3, and Hmob33 cDNAs as an Example of Human Brain-Specific Gene Studies

Brain-specific human genes were studied over the recent years in the Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics. Clones Hfb1, Hmob3, and Hmob33 were selected from human brain cDNA libraries by differential screening. The clones were sequenced, mapped, and tested for expression in various human tissues. In vitro and in silico experiments identified Hfb1 as an earlier unknown complexin 2 gene (CPLX2) fragment, which codes for the large 3"-untranslated region of the CPLX2 mRNA. Hmob3 proved to correspond to an earlier unknown fragment of the large 3"-untranslated region of the human MAP1B mRNA. With Hfb1 and Hmob3, new terminal exons were revealed and exact structures established for CPLX2 and MAP1B. Hmob33 was identified as a fragment of the 3"-terminal exon of a new gene, MOB, which codes for a yet unknown evolutionarily conserved transmembrane protein. The structure of the deduced protein product was analyzed.     

Structural and functional analyses of the Hfb1, Hmob3 and Hmob33 cDNAs as an example of human brain-specific gene studies

Brain-specific human genes were studied over the recent years in the Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics. Clones Hfb1, Hmob3, and Hmob33 were selected from human brain cDNA libraries by differential screening. The clones were sequenced, mapped, and tested for expression in various human tissues. In vitro and in silico experiments identified Hfb1 as an earlier unknown complexin 2 gene (CPLX2) fragment, which codes for the large 3'-untranslated region of the CPLX2 mRNA. Hmob3 proved to correspond to an earlier unknown fragment of the large 3'-untranslated region of the human MAP1B mRNA. With Hfb1 and Hmob3, new terminal exons were revealed and exact structures established for CPLX2 and MAP1B. Hmob33 was identified as a fragment of the 3'-terminal exon of a new gene, MOB, which codes for a thus far unknown evolutionarily conserved transmembrane protein. The structure of the deduced protein product was analyzed.     

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.     

Isolation and characteristics of clones complementary to mRNA of the murine cellular tumor antigen p53

43 cDNA clones specific for murine cellular tumor antigen p53 were isolated from a library constructed using 17S fraction of mRNA from the SV40 transformed murine fibroblasts (SVT2). These clones contain the whole coding region of p53 mRNA and the most of non-translated sequences. A plasmid containing 1.8 kb insert of p53 cDNA was constructed. The p53 specific insert in this plasmid was colinear with p53 mRNA, as revealed by S1 nuclease analysis. 5'-region of the p53 gene comprising non-translated and promoter areas was cloned from the mouse genomic library. A combined clone containing promoter and the whole region, corresponding to p53 mRNA has been constructed.     

Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.

Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.     

Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato

Differential screening of a cDNA bank constructed from ripe tomato fruit mRNA allowed the isolation of cDNA clone 2A11 which is entirely fruit-specific, is expressed at steadily increasing levels from anthesis to breaker, and accounts for approximately 1% of the messenger RNA in mature tomato fruit. A genomic clone corresponding to the 2A11 cDNA was isolated from a tomato genomic library. Sequence comparison of the cDNA clone with the genomic clone shows they are identical over the shared region with the genomic clone possessing a single large intron near the 5 end of the message.The open reading frame of 2A11 would encode a sulfur-rich polypeptide 96 amino acids in length. The identity of the putative protein is unknown. In situ hybridization shows that the 2A11 message is found throughout the pericarp cells in a tomato fruit. In contrast, in situ hybridization of early ripening stages with a polygalacturonase probe shows higher mRNA levels in cells of the outer pericarp and cells surrounding the vascular regions of the pericarp.     

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.