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.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Molecular cloning of cDNA coding for human preprourokinase

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, fractionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.     

Structure and expression of the human XPBC/ERCC-3 gene involved in DNA repair disorders xeroderma pigmentosum and Cockayne's syndrome

The human XPBC/ERCC-3 was cloned by virtue of its ability to correct the excision repair defect of UV-sensitive rodent mutants of complementation group 3. The gene appeared to be in addition implicated in the human, cancer prone repair disorder xeroderma pigmentosum group B, which is also associated with Cockayne's syndrome. Here we present the genomic architecture of the gene and its expression. The XPBC/ERCC-3 gene consists of at least 14 exons spread over approximately 45 kb. Notably, the donor splice site of the third exon contains a GC instead of the canonical GT dinucleotide. The promoter region, first exon and intron comprise a CpG island with several putative GC boxes. The promoter was confined to a region of 260 bp upstream of the presumed cap site and acts bidirectionally. Like the promoter of another excision repair gene, ERCC-1, it lacks classical promoter elements such as CAAT and TATA boxes, but it shares with ERCC-1 a hitherto unknown 12 nucleotide sequence element, preceding a polypyrimidine track. Despite the presence of (AU)-rich elements in the 3'-untranslated region, which are thought to be associated with short mRNA half-life actinomycin-D experiments indicate that the mRNA is very stable (t 1/2 greater than 3h). Southern blot analysis revealed the presence of XPBC/ERCC-3 cross-hybridizing fragments elsewhere in the genome, which may belong to a related gene.     

Sequence of the 3''-noncoding and adjacent coding regions of human gamma-globin mRNA.

In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present.     

Cloning, primary structure determination and expression of preproinsulin cDNA from human insulinoma in Escherichia coli]

A human insulinoma cDNA library was constructed in expression plasmid vector pUEX1. Clone pUEX1Ins12 was selected from human insulinoma cDNA library by means of hybridization with the insulin probe and a nucleotide sequence of the insertion was determined. It codes for full size amino acid sequence preproinsulin and furthermore, contains the entire 3'-end of noncoding mRNA region and 44 nucleotides from the 5'-untranslated region. The bacterial strain pUEX3Ins8 producing preproinsulin as beta-galactosidase fusion protein was constructed.     

Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase

The Escherichia coli asnS gene codes for asparaginyl-tRNA synthetase (NRSEC). We have sequenced the asnS region, including 382 bp of the 5'-untranslated region, 1398 bp of the coding region and 280 bp of the 3'-untranslated region. The DNA-derived NRSEC amino acid (aa) sequence was confirmed by direct aa sequencing of the N-terminal parts of the native protein and of a 28-kDa internal fragment generated by trypsin digestion. The asnS gene product has been purified to homogeneity using three chromatographic steps. Sequence comparison of the deduced NRSEC sequence with all aminoacyl-tRNA synthetase sequences showed significant homologies with the yeast aspartyl-tRNA synthetase and weaker relationships with other aminoacyl-tRNA synthetases for aa with an XAX codon.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Molecular Genetics and Genomics - The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the...     

Cloning and sequence analysis of porcine myoglobin cDNA

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.     

Cloning, sequencing, and characterization of alternatively spliced glutaredoxin 1 cDNA and its genomic gene: chromosomal localization, mrna stability, and origin of pseudogenes

Alternatively spliced human glutaredoxin (Grx1(as)) cDNA was isolated from a neutrophil cDNA library, using a (32)P-labeled human glutaredoxin (Grx1) cDNA probe under non-stringent conditions. The sequence of Grx1(as) cDNA indicated that the open reading frame of the gene was identical to the open reading frame of the previously reported first human glutaredoxin (Grx1) cDNA, but the 3'-untranslated region of Grx1(as) was not homologous to Grx1 cDNA. Northern blot and RT-PCR analyses showed Grx1(as) mRNA was expressed in normal human neutrophils and transformed cells including U937, HL-60, THP, and Jurkat cells. Cloning and sequencing of the genomic gene corresponding to Grx1(as) cDNA showed that two different glutaredoxin cDNAs (Grx1(as) and Grx1) were generated from the same genomic gene via alternative splicing. Origination of Grx1(as) and Grx1 from the same gene was confirmed by chromosomal localization of the Grx1(as) gene to chromosome 5q13, the same location where the Grx1 gene was localized previously. During screening of the Grx1(as) genomic gene, two additional glutaredoxin pseudogenes were also isolated. Surprisingly, these pseudogenes contained 3'-untranslated regions that were nearly identical to the 3'-untranslated regions of Grx1(as,) not Grx1, cDNA. Because 3'-untranslated regions may be important in stabilizing mRNAs, the effect of the two 3'-untranslated regions of Grx1 and Grx1(as) on mRNA stability was investigated using luciferase reporter vectors with the 3'-untranslated regions. Luciferase activity was 2.6-fold greater in cells transfected with the reporter vector containing the 3'-untranslated region of Grx1(as) cDNA compared with the 3'-untranslated region of Grx1 cDNA. These data indicate that Grx1(as) cDNA is an alternatively spliced human Grx1 cDNA and that the Grx1(as) 3'-untranslated region may have a role in stabilizing mRNA.     

Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3'-untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3'-untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.