Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.     

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.)

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.     

Cloning and sequence analysis reveal structural variation among related zein genes in maize

We have isolated a gene encoding one of the 19,000 dalton zein proteins from a maize genomic library constructed in Charon 4A. This gene occurs on a 7.7 kb Eco RI fragment, and based on Southern hybridization analysis, represents one of several homologous sequences present in the maize genome. The nucleotide sequence of the gene predicts a protein composed of 235 amino acids, including a signal peptide of 21 amino acids. There are no intervening sequences in the gene. By comparing the nucleotide sequence of this gene with that of a homologous cDNA clone, we have identified a basis for microheterogeneity within the gene family. The 5′ nucleotide sequences of the genomic and cDNA clones are identical, but they differ in the center of the protein, where repeated amino acid sequences occur. A nucleotide sequence encoding a conserved peptide of 20 amino acids is repeated nine times in the center of both of these clones.     

Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.

A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.     

新疆绵羊种布鲁氏菌HtrA基因的克隆与序列分析

根据已发表的牛流产型布鲁氏菌(B.abrotus)HtrA(High temperature requirment A)基因的核酸序列设计引物,从新疆绵羊种布鲁氏菌(B.ovis)基因组中扩增到了大约1600bp的片段。将该片段纯化后克隆到PBS-T载体上,对所得到的重组质粒进行PCR鉴定、酶切分析后,对克隆的片段进行测序表明,新疆绵羊种布鲁氏菌HtrA基因与发表的牛流产型布鲁氏菌、马耳他布鲁氏菌(B.melitensis)、猪种布鲁氏菌(B.suis)的HtrA基因核苷酸序列的同源性分别为99.68%、99.81%、99.55%,推导的氨基酸序列也存在很高的同源性。     

Cloning and characterization of a gene encoding the endopolygalacturonase of Penicillium griseoroseum

A conserved region of a polygalacturonase (PG) gene from Penicillium griseoroseum was PCR amplified and used to screen a genomic library from this fungus. The nucleotide sequence of the isolated clone (pggI) consisted of 1497 bp, including a coding region of 1251 bp. This region potentially encodes a protein of 376 amino acids, and is interrupted by two introns. Extensive homology was observed between this protein and several fungal endopolygalacturonases. DNA hybridization analyses revealed that there is a low copy number of pggI in the P. griseoroseum genome, probably one or two copies.     

Analysis of OPT8511 RAPD fragments closely linked with cold sensitivity at seedling stage in rice (Oryza sativa L.)

OPT8(511) was confirmed to be strongly associated with cold sensitivity of rice by random amplified polymorphic DNA (RAPD) analysis for the cold tolerance with 94 F2 population crossed with 'Dular' (cold sensitive cultivar) and 'Toyohatamochi' (cold resistant cultivar). A DNA marker from the RAPD fragment, OPT8(511), has been cloned with genomic DNA from rice cultivar ('Dular') and the nucleotide sequence has been determined. The nucleotide sequence revealed that the putative open reading frame was 511 base pairs and contained 169 amino acid residues. It is 79% and 57% identical to the rice cDNA (C26347) in DataBank at the nucleotide and amino acid sequence levels, respectively. The clone OPT8(511) specifically amplified a 511 bp band from the DNA of cold sensitive cultivars. Use of this marker could facilitate early selection of character associated with cold tolerance in rice.     

Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.     

中国人SRY基因的分离、克隆和核苷酸序列分析

睾丸决定因子基因(Testis-determining factor,TDF)位于Y染色体短臂上,它的表达产物诱导睾丸组织的发生。SRY基因(Sex-determining Region of the Y)位于Y染色体的性别决定区内,许多特征显示SRY就是TDF。我们选用与SRY基因相应的引物,用PCR技术对正常人男女各10例的基因组DNA进行扩增。将特异扩增的男性SRY基因片段重组到质粒PUC12中,得到含有中国人SRY基因片段的克隆,命名为PSY-1、PSY-2。用[~(32)p]标记重组质粒中的SRY基因片段作探针,与PCR结果进行Southern杂交,男性样品均显示特异杂交带,女性样品为阴性。用末端终止法测定克隆的SRY基因片段的全部核苷酸序列为299bp,含有SRY基因中高度保守及功能特异性区域的240bp,我们对此进行了讨论。     

Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp Eco RI/ Xba I fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb Bam HI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

杜氏盐藻叶绿体atpA基因片段的克隆及序列分析

根据莱因衣藻、卵形肾藻、普通小球藻等10种藻类的atpA全基因氨基酸高度保守序列,设计简并引物,利用PCR方法从盐藻叶绿体DNA中扩增出约400bp的片段,将该片段连接到T-vector上进行序列测定。结果表明,核苷酸长度为405bp,编码135个氨基酸。推导的氨基酸序列与莱因衣藻的同源性为92%,普通小球藻88%,Mesostigmaviride87%,卵形肾藻86%,Cyanidioschyzonmerolae85%。以所克隆的DNA片段为探针,与盐藻叶绿体基因组进行SouthernBlot杂交结果有明显的杂交信号。据此可推断本实验中所克隆的序列为杜氏盐藻叶绿体atpA基因片段。该基因序列已被GenBank收录,接受号为AY435096。     

A partial cDNA clone for porcine glucosephosphate isomerase: isolation, characterization and use in detection of restriction fragment length polymorphisms

A cDNA library for porcine skeletal muscle was established in the vector pBR322. The library was screened with an oligonucleotide probe coding for a hexapeptide from glucosephosphate isomerase (Gpi). A positive clone with an insert of about 450 bp and restriction sites for PstI, BamHI and PvuII was isolated. A 362-bp PstI fragment was sequenced and shown to contain the codons for the hexapeptide as well as the remaining 29 amino acids of this Gpi peptide. The PstI fragment was used to probe pig genomic DNA. The restriction enzymes PvuII and SacI detected a set of polymorphisms with five bands, behaving as a set of insertion/deletion polymorphisms.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.