Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.     

Deficiency of human complement protein C4 due to identical frameshift mutations in the C4A and C4B genes

The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations is both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.     

The amino terminal sequences of bovine and human chromogranin A and secretory protein I are identical

The amino terminal sequences of bovine and human adrenal medullary chromogranin A have been determined. Their sequences are identical and also identical to the published sequence of secretory protein I from the parathyroid gland. This data indicates that the previously published sequence of chromogranin A is incorrect at residues 2 and 19. These data confirm earlier observations of a substantial similarity between secretory protein I and chromogranin A and, in fact, strongly suggest that they are identical.     

SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp

Genetic studies in shrimp have focused on disease, with production traits such as growth left unexamined. Two shrimp species, Litopenaeus vannamei and Penaeus monodon, which represent the majority of US shrimp imports, were selected for single nucleotide polymorphism (SNP) discovery in alpha-amylase (AMY2) and cathepsin-l (CTSL), both candidate genes for growth. In L. vannamei, four SNPs were found in AMY2 and one SNP was found in CTSL. In P. monodon, one SNP was identified in CTSL. The CTSL gene was mapped to linkage group 28 of P. monodon using the female map developed with the Australian P. monodon mapping population. Association analyses for the AMY2 and CTSL genes with body weight (BW) were performed in two L. vannamei populations. While neither gene was found to be significantly associated with BW in these populations, there was a trend in one population towards higher BW for allele G of CTSL SNP C681G.     

Characterization of mouse and human B7-H3 genes

T cell activation and immune function are regulated by costimulatory molecules of the B7 superfamily. Human B7-H3 is a recent addition to this family and has been shown to mediate T cell proliferation and IFN-gamma production. In this work we describe the identification of the mouse B7-H3 homolog, which is ubiquitously expressed in a variety of tissues. Activated CD4 and CD8 T cells express a putative receptor that can be recognized by soluble mouse B7-H3-Ig molecules. While the mouse B7-H3 gene was found to contain a single copy, we discovered a novel isoform of human B7-H3 (named as B7-H3b hereafter) with four Ig-like domains that results from gene duplication and differential splicing. B7-H3b is the major isoform expressed in several tissues. This structural information suggests a genetic variation of the B7-H3 gene in mammalian species.     

ATAD 3A and ATAD 3B are distal 1p-located genes differentially expressed in human glioma cell lines and present in vitro anti-oncogenic and chemoresistant properties

Human oligodendrogliomas are chemosensitive gliomas usually characterized by a loss of heterozygosity in the large distal regions of the short arm of chromosome 1 (1p LOH). Chemoresistant astrocytomas do not have this genetic signature, suggesting that the 1p arms may contain anti-oncogene and/or genes enabling chemoresistance. We have focused here on two human 1p-distal genes, ATAD 3A and ATAD 3B (1p36-33), and analyzed their gene products in normal human cell lines and tissues and in glioma-derived human cell lines. Using specific anti-peptide antibodies, we have found that ATAD 3A is ubiquitously expressed, whereas ATAD 3B is expressed in embryonic tissues, adult germinative zone and in astrocytoma cell lines but it is not expressed in oligodendroglioma cell lines or in the adult cortex. Furthermore, we have found that human glioma cell lines overexpressing or underexpressing ATAD 3A and ATAD 3B, show modified cell growth, anchorage-independent growth, and chemoresistance to doxorubicin and other genotoxic drugs. These results demonstrate the potential for ATAD 3B as a putative marker in discriminating astrocytomas from oligodendrogliomas. We also have shown that the loss of ATAD 3A/3B may be involved in the transformation pathway and the chemosensitivity of oligodendrogliomas.     

Two non-allelic human interferon alpha genes with identical coding regions

A hitherto undescribed human interferon-alpha (IFN-alpha) gene, IFN-alpha 13, has been isolated and characterized. Its entire coding sequence is completely identical with that of IFN-alpha 1; the 5' and 3' non-coding regions differ by 4.5% and 3.8%, respectively. As the two genes are not allelic, we conclude that the similarity is due to a very recent gene conversion event. Both genes are expressed in virus-induced leukocytes.     

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.     

Ly-6A.2 and Ly-6E.1 molecules are antithetical and identical to MALA-1

Rat monoclonal antibodies YE3/19.1, defining the murine-activated lymphocyte antigen MALA-1, and D7, detecting an Ly-6 locus-controlled antigen, bound highly purified Ly-6E.1. On western blots of lymphocyte surface proteins which had been solubilized and electrophoretically separated in octylglucoside, they detected bands which comigrated with Ly-6A.2 or Ly-6E.1 antigens. On cells or in an immunoassay they blocked alloantibodies against Ly-6A.2 or Ly-6E.1. The tissue distribution of MALA-1 also correlated with Ly-6A.2 or Ly-6E.1. Upon octylglucoside or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these antigens displayed similar sizes. Thus, Ly-6A.2 and Ly-6E.1 are most likely products of alternate alleles. Electrophoretic analysis showed a similar size and charge for Ly-6A.2, Ly-6B.2, Ly-613.2, and Ly-27.2. Ly-6C.2 and Ly-28.2 appeared to be identical, and were similar in size to Ly-6A.2, but they differed in charge and in intrachain disulfide constraints. Since Ly-613.2 and Ly-27.2 may represent the same or different epitopes on the Ly-6A.2 molecule, the previously postulated five Ly-6-like antigens that were thought to be separable on the basis of tissue distribution, may represent no more than three separate proteins which can be assigned to one of two distinct categories by electrophoretic mobility in gels containing octylglucoside.     

Mutations in both the 2B and 2C genes of hepatitis A virus are involved in adaptation to growth in cell culture.

Oligonucleotide-directed mutagenesis of an infectious cDNA clone of wild-type hepatitis A virus was performed to determine which mutations acquired in the nonstructural 2B and 2C genes during adaptation to growth in cell culture were effective in enhancing virus growth in vitro. Results of transfection assays demonstrated that one mutation in the 2B gene and two mutations in the 2C gene were responsible for an increased efficiency in growth, but growth enhancement required the participation of at least two of the three mutations.     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

The human desmin and vimentin genes are located on different chromosomes

We have used somatic cell hybrids of Chinese hamster X man and mouse X man to localize the genes (des and vim) encoding the intermediate filaments desmin and vimentin in the human genome. Southern blots of DNA prepared from each cell line were screened with hamster cDNA probes specific for des and vim genes, respectively. The single-copy human des gene is located on chromosome 2, and the single-copy human vim gene is assigned to chromosome 10. Partial restriction maps of the two human genomic loci are presented. A possible correlation of the des locus with several reported hereditary myopathies is discussed.