Transient occurrence of extrachromosomal DNA of an Arabidopsis thaliana transposon-like element, Tat1.

Analysis of 11 genomic clones containing the S-adenosylmethionine synthetase 1 gene (sam1) of Arabidopsis thaliana revealed the presence of a 431-base-pair (bp) insertion in the 3' end of sam1 in one of these clones. The inserted sequence, called Tat1, shows structural features of a transposon. It is flanked by a 5-bp duplication of the target site DNA and has 13-bp inverted repeats at its termini. Two highly homologous elements situated in a different genomic context were isolated from a genomic library. Genomic Southern analysis indicates that there are at least four copies of Tat1 present in the A. thaliana ecotype Columbia genome. Different hybridization patterns are observed with DNAs derived from different ecotypes of Arabidopsis thaliana, indicating that the element has moved since the divergence of these ecotypes. In two populations of A. thaliana, linear extrachromosomal Tat1-homologous DNA has been observed. The presented data are consistent with the hypothesis that Tat1 is an active transposable element.     

Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene.

Several plasmid-encoded beta-lactamases are on multiresistance transposable elements. The OXA-1 beta-lactamase gene is part of Tn2603, which is borne on the R plasmid RGN238. We report here the complete nucleotide sequence of the OXA-1 beta-lactamase gene and flanking sequences. The OXA-1 gene shows a greater than 50% sequence divergence from the OXA-2 gene, yet there is significant functional similarity at the peptide level. Analysis of 5' and 3' flanking sequences shows that Tn2603 differs from its probable precursor, Tn21, by a precise 1004-base-pair insertion, containing the OXA-1 structural gene, at the target sequence AAAGTT, which is located between the Tn21 streptomycin/spectinomycin (aadA) promoter and its structural gene. A 5- for 6-base repeat of the target sequence is found at the end of the insertion. The same precise insertion and repeat of the target sequence are found for the OXA-2 gene from R46. The 5' flanking regions of two other genes, the trimethoprim-resistance gene from R388 and the gentamicin resistance (aadB) gene from pDGO100, are greater than 98% homologous to the 5' flanking sequences of the OXA-1, OXA-2, and aadA genes until they diverge at the target sequence. From the available sequence data a recombinational hot spot is defined at the nucleotide level 5' of the aadA gene of Tn21, and a second potential hot spot is proposed 3' of this gene.     

Gene movement by Helitron transposons contributes to the haplotype variability of maize

Different maize inbred lines are polymorphic for the presence or absence of genic sequences at various allelic chromosomal locations. In the bz genomic region, located in 9S, sequences homologous to four different genes from rice and Arabidopsis are present in line McC but absent from line B73. It is shown here that this apparent intraspecific violation of genetic colinearity arises from the movement of genes or gene fragments by Helitrons, a recently discovered class of eukaryotic transposons. Two Helitrons, HelA and HelB, account for all of the genic differences distinguishing the two bz locus haplotypes. HelA is 5.9 kb long and contains sequences for three of the four genes found only in the McC bz genomic region. A nearly identical copy of HelA was isolated from a 5S chromosomal location in B73. Both the 9S and 5S sites appear to be polymorphic in maize, suggesting that these Helitrons have been active recently. Helitrons lack the strong predictive terminal features of other transposons, so the definition of their ends is greatly facilitated by the identification of their vacant sites in Helitron-minus lines. The ends of the 2.7-kb HelB Helitron were discerned from a comparison of the McC haplotype sequence with that of yet a third line, Mo17, because the HelB vacant site is deleted in B73. Maize Helitrons resemble rice Pack-MULEs in their ability to capture genes or gene fragments from several loci and move them around the genome, features that confer on them a potential role in gene evolution.     

Insertion of a repetitive element at the same position in the 5''-flanking regions of two dissimilar yeast tRNA genes.

The regions 5' proximal to many yeast tRNA genes exhibit a high frequency of DNA sequence polymorphisms. DNA sequence analysis of polymorphic variants of SUQ5, a tRNA Ser UCA gene, and SUP2, a tRNA Tyr gene, shows that in each case one sequence variant of the tRNA gene is 346 base pairs longer than the other. The longer variants appear to have arisen from the shorter ones by the insertion of nearly identical copies of a 341-base pair sigma element into a site 16 base pairs upstream from the 5' ends of the tRNA-coding regions. The sequences of the two copies of the sigma element differ at only five positions. The element has a number of properties that are typical of many transposable elements: (i) there is a perfect eight-base-pair inverted repeat at its ends, (ii) these ends are flanked by a five-base-pair direct repeat of a sequence that occurs only once in the target DNA, (iii) there are approximately 20 copies of the element in the yeast genome, and (iv) there is considerable strain-to-strain variation in the sizes of the restriction fragments on which these copies lie. The presence of the sigma element has no gross effect on the phenotype of a SUP2 ochre suppressor. Analysis of the SUQ5 and SUP2 sequences favors the hypothesis that sigma is a transposable element with a novel type of insertion specificity, which is primarily based on the presence of a tRNA-coding region a fixed distance from the insertion site, rather than on the immediate target sequences.     

Intronless human dihydrofolate reductase genes are derived from processed RNA molecules.

Three groups of recombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) were isolated from two human DNA clone libraries. One recombinant (lambda hDHFR-1) contains three exons that encode the COOH-terminal portion of human DHFR. The other two human DHFR genes (hDHFR-psi 1 and hDHRF-psi 2) lack introns. hDHFR-psi 2 contains several in-phase termination codons and is only 93% homologous to the normal human DHFR coding sequences, whereas hDHFR-psi 1 has an open reading frame and is virtually identical to the coding sequence of the normal DHFR gene. The region of DNA sequence homology between each intronless gene and the normal DHFR gene extends 2.9 kilobases beyond the end of the coding sequences. At the 3' end of this homologous sequence, each intronless gene has an A-rich tract. The lack of introns and the presence of the 3' A-rich tract suggest that hDHFR-psi 1 and hDHFR-psi 2 were derived from processed RNA molecules. A short DNA sequence, 60 nucleotides 5' to the ATG start codon in lambda hDHFR-psi 2, is directly repeated immediately after the 3' A-rich tract; such terminal direct repeats also flank integrated proretroviruses and transposable DNA elements and are thought to be the hallmark of inserted DNA sequences.     

tau, a repeated DNA sequence in yeast.

We have found a 371-base-pair (bp) repeated DNA element, tau, in Saccharomyces cerevisiae. The ends of tau are composed of a 5-bp inverted repeat, similar in sequence to those reported for the Ty, sigma, copia, and spleen necrosis virus elements. These inverted repeats are flanked by 5-bp direct repeats of a target sequence that occurs only once in an allele that lacks the tau element. This overall structure is characteristic of transposable elements. Like sigma, tau elements have been found (in both orientations) closely associated with tRNA genes (409 and 198 bp from the 5' end, respectively). It is noteworthy that one representative of tau was isolated in a concentric insertion of tau, delta, and sigma.     

弓形虫P24基因敲除转染质粒pGB/P5-P3的构建

目的构建弓形虫P24(TgP24)基因敲除转染质粒pGB/P5P3(GRA2/BleP245’UTRP243,UTR),为TgP24基因敲除奠定基础。方法根据TgP24基因序列,设计并合成两对特异引物(P1toP4),采用PCR技术特异扩增TgP24基因的5,端非翻译区2.5kb片段(P245’UTR)和3’端非翻译区2.89kb片段(P243’UTR),将其分别亚克隆入pCR2.1TOPOTA载体,构建质粒P245’UTR/TA和P243’UTR/TA;重组质粒P245’UTR/TA经KpnⅠ和BglⅡ双酶切后,再将纯化的P245’UTR片段亚克隆入转染质粒GRA2/Ble的KpnⅠ和BglⅡ位点,构建重组质粒pGBP5(GRA2/BleP245’UTR);重组质粒P243’UTR/TA经BamHⅠ和NotⅠ双酶切后,纯化P243’UTR片段,再将其定向克隆到重组质粒pGBP5的BamHⅠ和NotⅠ位点,从而构建弓形虫TgP24基因敲除转染质粒pGB/P5P3。重组质粒经DNA序列测定证实目的片段插入正确。结果经过PCR筛选、限制性酶切及DNA测序鉴定,证实P245’UTR和P243’UTR两片段正确插入质粒GRA2/Ble的KpnⅠ和BglⅡ及BamHⅠ和NotⅠ位点,位于药物选择ble基因的上,下游。结论成功构建弓形虫P24基因敲除转染质粒pGB/P5P3。     

DNA methylation patterns of the gamma delta beta-globin genes in human fetal and adult erythroid tissues.

An investigation of the correlation between the gamma----beta-globin switch and DNA methylation was carried out. The restriction patterns obtained with methylation-sensitive and -insensitive enzymes indicated hypomethylation in the promoter region of the gamma-globin genes in fetal liver DNA but high methylation of the same region in all other samples (except in the presence of an elevated erythroblast count or leukemia). All samples appeared to be partially hypomethylated at the 5' end of the delta-globin gene and hypomethylated at the 3' region of the beta-globin gene. Although consistent with a role for DNA methylation in globin gene regulation, the results also suggest that other factors besides methylation may be required for regulation of the level of expression, and switching of the globin genes.     

Isolation and characterization of rat skeletal muscle and cytoplasmic actin genes.

Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences. By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene. Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries. One of them, Act 15, contains the skeletal muscle actin gene. Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene. Both genes have a large intron very close to the 5' end of their transcribed region, followed by several small introns. DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness. Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others.