Transposable elements associated with constitutive expression of yeast alcohol dehydrogenase II

The yeast structural gene ADR2, coding for the glucose-repressible alcohol dehydrogenase (ADHII), has been isolated by complementation of function in transformed yeast. The chromosomal DNA from nine yeast strains with cis-dominant constitutive mutations (ADR3^c) has been investigated by restriction enzyme analysis, using the cloned ADR2 DNA as a hybridization probe. Seven mutants appear to have insertions of approximately 5.6 kb near the 5′ end of the ADR2-coding region. Four of these insertions have the same restriction pattern as the yeast transposable element Tyl. Two differ from Tyl by the presence of an additional Hind III site, and a seventh insertion differs from Tyl at a number of restriction sites. All are inserted in the same orientation with respect to the structural gene. A DNA fragment containing the ADR2 gene and adjacent sequences from a constitutive mutant has been cloned and shown by heteroduplex analysis to contain an insertion near the 5′ end of the structural gene. The cloned insertion sequence hybridizes to multiple genomic DNA fragments, indicating that it contains a moderately repetitive sequence. Thus it appears that insertion of a transposable element near the 5′ terminus of the structural gene can produce constitutive expression of a normally glucose-repressed enzyme. Such insertions seem to be the most common way of generating cis-dominant constitutive mutations of ADHII.     

Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species

A collection of lethal and semi-lethal P-element insertions in the 70CD region of chromosome 3 of Drosophila melanogaster was used to investigate genes and gene arrangements by a combination of genetic, cytological, functional and molecular methods. The 12 lethal insertions studied fall into seven complementation groups of six genes. Lethal phases, expression patterns and other phenotypic aspects of these genes were determined. The genes and additional available sequences were placed on cloned genomic DNA fragments and arranged in an EcoRI map of 150kb that covers approximately the bands 70C7-8 to 70D1. Determination of deficiency breakpoints links the genetic, physical and molecular data. The sequences adjacent to seven independent P-element insertions were established after plasmid rescue or polymerase chain reaction. Similarity searches allowed the assignment of the P-element insertions to known mutations, expressed sequence tags, sequence tagged sites, or homologous genes of other species. Among these were identified a putative transacylase, a putative cell cycle gene, and the gene responsible for the dominant Polycomb-suppressor phenotype of devenir. The genomic sequence of the l(3)70Ca/b gene reveals a novel heat shock protein (hsc70Cb). l(3)70Da was identified as a member of the CDC48/PEX1 ATPase family and its coding sequence was determined.     

Ruminant globin gene structures suggest an evolutionary role for Alu-type repeats

Bovine fetal and adult globin genes were cloned and subjected to DNA sequence analysis. Both of these genes contained insertions of Alu-type repetitive DNA within their introns. Comparison of cow and goat beta-type globin genes indicates that intragenic DNA insertions played a role in their evolution. These data support the theory that Alu-type repeats maintain genetic diversity by inhibiting gene conversion.     

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids.

Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.     

Simple method of cloning eukaryote DNA: production of certain new ribosomal genes of Drosophila

We propose a simple method which allows to receive a collection of clones containing recombinant plasmids. It is based on the ligation of the longer fragment of pBR332 formed by EcoRI and BamH1 with eukaryotic DNA (from Drosophila melanogaster embryo in this case) partially cleaved with EcoRI and BamHI. This approach gave us 10(4) colonies from 1 microgram of Drosophila DNA and 0.1 microgram of the BamHI--EcoRI "vector". About 0.5% of all clones carried the fragments of ribosomal genes with insertions in the 26S gene. Ribosomal genes lacking insertions did not enter the collection due to some peculiarities in their restriction map. The sites of cleavage are mapped in eight recombinant plasmide for HindIII, BamHI and EcoRI. These maps show that some insertions within 26S gene have not been cloned earlier. The mean length of cloned fragments is 11.8 kilobases, the mean number of EcoRI and BamHI restriction sites are 1.2 and 1.0, respectively. The electrophoretical screening of plasmids using cetyl trimethyl ammonium bromide was developed.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization.

Multiple nitrocellulose DNA filter replicas of plaques of in vitro generated recombinants of phage lambda and Saccharomyces cerevisiae have been screened by hybridization with 32P-labeled cDNA probes. These probes were representative of total poly(A)-containing RNA of yeast cells grown on acetate, galactose, glucose or maltose. This approach allows the use of specific differences in total RNA populations as probes for gene isolation. Five "galactose-induced" clones have been isolated. Expression of the RNA coding regions on at least two cloned sequences, Sc481 and Sc482, is regulated by genes known to control the expression of the structural genes required for the conversion of exogenous galactose to endogenous glucose-1-phosphate. One cloned sequence, Sc484, is expressed during growth on all carbon sources except glucose, and is not under control by the galactose regulatory genes. This clone contains a sequence that is repeated 3 times in the yeast genome. The cloned fragment Sc481 contains coding regions for all or part of three galactose"induced RNAs and may correspond to the GAL 1, GAL 7, GAL 10 gene cluster region of chromosome II.     

Cloning, sequence and expression of the lactate dehydrogenase gene from the human malaria parasite, Plasmodium vivax

Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria. To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax ( PvLDH) Belem strain. The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E. coli. Structural differences between human LDHs and PfLDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs. The sequence similarity between PvLDH and PfLDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.     

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术,从括楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明,克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％,９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９     

The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12

The gene xylE, coding for xylose-proton symport in Escherichia coli, was cloned and its DNA sequence determined. The cloning strategy utilized lambda placMu insertions and exploited the proximity of xylE to malB. A 2.8-kilobase HincII fragment of cloned DNA restored [14C]xylose transport and xylose-proton symport activities to a xylose transport-negative strain. The xylE gene was identified as a 1473-base pair open reading frame, located 373 base pairs downstream of malG, encoding a hydrophobic protein of Mr 53,607. The amino acid sequence of XylE bore little resemblance to the lactose-proton LacY symporter or melibiose-sodium MelB symporter, but a high degree of homology was found with the arabinose-proton AraE symporter of E. coli and glucose transport proteins of mammals. Structural analyses and comparisons suggest that 12 membrane-spanning segments may occur in the XylE protein.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

A broad-host-range cloning vector transposable to various replicons

A system is described for the stable insertion of cloned DNA sequences into the chromosomes of Gram-negative bacteria. Two broad-host-range plasmids form the basis of the system: one (the "carrier") contains a transposable DNA sequence into which foreign DNA can be cloned; the second (the "helper") provides transposition functions in trans. Both plasmids can be readily transferred between Gram-negative bacteria by conjugation. Instability of the carrier allows enrichment for the products of transposition to the chromosome of the new host, following which the insertion can be stabilised by elimination of the helper. The system was successfully tested in Escherichia coli, Methylophilus methylotrophus and Pseudomonas aeruginosa, and the insertions were stable in each case (less than 0.02% loss per generation).     

DNA sequence of the white locus of Drosophila melanogaster

The DNA sequence of the white locus of Drosophila melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. We also report the sequence of a complementary DNA clone which established the position of the 3' end of the white RNA on this genomic sequence. The probable exon-intron structure of the gene has been predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with seven insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wzm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11)), one deletion mutant (white-spotted 4 (wsp4)) and one internal duplication mutant (white-ivory (wi)) have been determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.