Genomic organization of the human COL3A1 and COL5A2 genes: COL5A2 has evolved differently than the other minor fibrillar collagen genes.

We report here on the complete structure of the human COL3A1 and COL5A2 genes. Collagens III and V, together with collagens I, II and XI make up the group of fibrillar collagens, all of which share a similar structure and function; however, despite the similar size of the major triple-helical domain, the number of exons coding for the domain differs between the genes for the major fibrillar collagens characterized so far (I, II, and III) and the minor ones (V and XI). The main triple-helical domain being encoded by 49-50 exons, including the junction exons, in the COL5A1, COL11A1 and COL11A2 genes, but by 43-44 exons in the genes for the major fibrillar collagens. Characterization of the genomic structure of the COL3A1 gene confirmed its association with the major fibrillar collagen genes, but surprisingly, the genomic organization of the COL5A2 gene was found to be similar to that of the COL3A1 gene. We also confirmed that the two genes are located in tail-to-tail orientation with an intergenic distance of approximately 22 kb. Phylogenetic analysis suggested that they have evolved from a common ancestor gene. Analysis of the genomic sequences identified a novel single nucleotide polymorphism and a novel dinucleotide repeat. These polymorphisms should be useful for linkage analysis of the Ehlers-Danlos syndrome and related disorders.     

Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants

Saccharomyces cerevisiae contains three genes that encode members of the histone H2A gene family. The last of these to be discovered, HTZ1 (also known as HTA3), encodes a member of the highly conserved H2A.Z class of histones. Little is known about how its in vivo function compares with that of the better studied genes (HTA1 and HTA2) encoding the two major H2As. We show here that, while the HTZ1 gene encoding H2A.Z is not essential in budding yeast, its disruption results in slow growth and formamide sensitivity. Using plasmid shuffle experiments, we show that the major H2A genes cannot provide the function of HTZ1 and the HTZ1 gene cannot provide the essential function of the genes encoding the major H2As. We also demonstrate for the first time that H2A.Z genes are functionally conserved by showing that the gene encoding the H2A.Z variant of the ciliated protozoan TETRAHYMENA: thermophila is able to rescue the phenotypes associated with disruption of the yeast HTZ1 gene. Thus, the functions of H2A.Z are distinct from those of the major H2As and are highly conserved.     

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.     

Characterization of ypa1 and ypa2, the Schizosaccharomyces pombe orthologs of the peptidyl proyl isomerases that activate PP2A, reveals a role for Ypa2p in the regulation of cytokinesis

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis. Cdc7p is the first kinase in the core SIN; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologs of mammalian phosphotyrosyl phosphatase activator. We have characterized this gene and its ortholog SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of protein phosphatase type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major protein phosphatase type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.     

甘蓝型油菜含油量的主基因+多基因遗传效应分析

应用多世代联合分析数量性状主基因和多基因混合遗传的统计方法,分析了甘蓝型油菜两个组合的5个世代——亲本P1、P2、F1、F2和F2：3家系材料含油量的遗传效应。结果表明,分离世代F2及F2：3家系含油量次数分布均呈混合的正态分布,符合主基因＋多基因的遗传特征。D-2模型是该项研究两个甘蓝型油菜杂交组合含油量的最适遗传模型,含油量的遗传是由一对加性主基因和加-显性多基因共同控制的。组合1（1141Bx垦C1-1）主基因加性效应值为-1．74,表明亲本1141B中主基因位点上的等位基因降低含油量,而亲本垦C1-1中的等位基因增加含油量。多基因加性效应值和显性效应值分别为1．20和-1．93;F2的主基因遗传力和多基因遗传力分别为68．21％和27．17％;F2：3的主基因遗传力和多基因遗传力分别为81．70％和16．80％。组合2（32Bx垦C1-2）主基因加性效应值为-3．74,表明亲本32B中主基因位点上的等位基因降低含油量,而亲本垦C1-2中的等位基因增加含油量。多基因加性效应值和显性效应值分别为-1．99和0．93;F2的主基因遗传力和多基因遗传力分别为66．20％,和28．10％;F2：3的主基因遗传力和多基因遗传力为81．00％和14．90％。两组合在F2：3家系世代含油量的主基因遗传力均较F2高,因此认为高含油量育种中在F2：3家系进行选择效率较高。     

A major gene for time of flowering in chickpea

A major gene for the number of days from sowing to appearance of the first flower (time of flowering) was identified in a cross between an extrashort duration chickpea (Cicer arietinum L.) variety, ICCV 2, and a medium duration variety, JG 62. The F2 population was advanced through the single-seed-descent method to develop random recombinant inbred lines (RILs). Time of flowering was recorded for the parents and 66 F(6) RILs from this cross that were grown in a Vertisol field in the post-rainy season of 1996-1997. Similarly the parents, F(1) and F(10) RILs were evaluated in 1997-1998. The F(1) flowered along with JG 62. The time of flowering for the two sets of RILs showed bimodal distributions with nearly equal peaks. One peak corresponded with ICCV 2 and the other with JG 62. This suggests that a single gene controls the difference for the time of flowering between ICCV 2 and JG 62 and the allele carried by the latter parent is dominant. To our knowledge no gene has been identified for the time of flowering in chickpea. Therefore the allele carried by JG 62 is designated as Efl-1 and that by ICCV 2 as efl-1. The proposed genotype for ICCV 2 is efl-1 efl-1 and for JG 62 is Efl-1 Efl-1. The genotype efl-1 efl-1 reduces the time of flowering at ICRISAT by nearly 3 weeks. The significance of this gene for breeding for early maturity and genome mapping has been discussed.     

Induction of human UGT1A1 by a complex of dexamethasone-GR dependent on proximal site and independent of PBREM

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role to conjugate bilirubin and prevent jaundice. There are two major elements for the induction of UGT1A1, such as PBREM (-3483/-3194), far from the promoter site, and HNF1 (-75/-63), near to the promoter site. In a previous report, we showed that the proximal HNF1 site is essential for the induction of UGT1A1 by glucocorticoid receptor (GR). In this report, we investigated the influence of PBREM on the induction of the UGT1A1 reporter gene by GR and PXR with dexamethasone (DEX). We confirmed that GR was transferred from cytosol into the nucleus in 15-30 min by DEX stimulation, but HNF1 was not. We constructed a reporter gene containing PBREM to compare the induction of the reporter gene without PBREM by DEX-GR. The results show that induction of the reporter gene with PBREM by DEX at 100 muM is the same level as the induction of the reporter gene without PBREM, although PBREM contains GRE. Co-transfection of hGR with the reporter gene did not show any influence of the induction of the reporter gene between the vector with and without PBREM. Meanwhile, by co-transfection of hPXR, the induction of the reporter gene with PBREM was significantly more than the induction of the reporter gene without PBREM at 100 muM DEX. This supports that hPXR induced UGT1A1 through PBREM by DEX. These results showed that PBREM has no relation with the induction by DEX-GR but the proximal site of UGT1A1 may function in stimulation by DEX-GR.     

Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver-specific enhancer for the human alpha-1-microglobulin/bikunin precursor.

Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4.     

Cloning and enhanced expression of the cytochrome P450nor gene (nicA; CYP55A5) encoding nitric oxide reductase from Aspergillus oryzae

We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.