GNP1, the high-affinity glutamine permease of S. cerevisiae

  Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have isolated the gene encoding the high-affinity glutamine permease and named it GNP1. The amino-acid sequence of GNP1, and its hydropathy profile of 12 transmembrane domains, closely resemble those of known amino-acid permeases. The Km of GNP1 for glutamine uptake was determined to be 0.59 mM. Cells lacking GNP1 exhibit reduced levels of glutamine transport, and are resistant to a toxic analog of glutamine, L-glutamic acid γ-monohydroxamate. Unlike other amino-acid permeases, whose expression is nitrogen-source limited, GNP1 is expressed on both rich and poor nitrogen sources. Received: 27 February / 30 March 1996     

汉坦病毒A9株L片段部分核苷酸序列分析

目的 测定我国分离的汉坦病毒Ａ９株Ｌ片段的部分核苷酸序列。方法 用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术扩增我国分离的汉坦病毒Ａ９株部分Ｌ片段ｃＤＮＡ,测定ＰＣＲ产物的核苷酸序列。结果 ＰＣＲ扩增产物为Ｌ片段４００６ｎｔ－４４５４１ｎｔ（根据７６－１１８株序列）,和已发表的汉坦病毒Ｌ片段序列比较,Ａ９株和汉滩病毒７６－１１８株同源性最高,为８５．２％,和其他不同型别汉坦病毒代表株ＨＲ８０－３９、     

汉坦病毒Q32株L和M片段全基因序列分析

目的测定汉坦病毒Q32株L、M片段的全长序列,了解其分子基础。方法设计特异的PCR引物,用RT-PCR技术分段扩增Q32株全长L、M片段,PCR产物纯化后直接测序,或用T-A克隆方法进行PCR产物克隆,然后进行遗传进化分析。结果Q32株L片段的全基因序列共6533个核苷酸,GC含量为37·38%,AT含量为62·62%,包含一个单一的开放读码框架(ORF),该读码框架从38到6493,由6456个核苷酸组成,编码2151个氨基酸,可以合成相对分子质量为2·46×105的蛋白。M片段全基因序列共3616个核苷酸,GC含量为39·44%,AT含量为60·56%,包含一个单一的开放读码框架(ORF),其读码框架从41到3448,由3408个核苷酸组成,编码1135个氨基酸,可以合成相对分子质量为1·26×105的蛋白。Q32编码的RNA聚合酶也有6个比较保守的区域。结论汉坦病毒Q32株M和L片段具有和其他汉坦病毒糖蛋白和RNA聚合酶相似的核苷酸一级结构,核苷酸序列虽有差异,但在氨基酸水平上的同源性仍很高。     

5株呼吸道合胞病毒地方株F蛋白基因序列分析

目的呼吸道合胞病毒（ＲＳＶ）Ｆ蛋白是ＲＳＶ感染免疫中最重要的病毒蛋白，为了解我国ＲＳＶ地方株Ｆ蛋白的基因状况和变异特征，随机选取北京、广州、长春和河北四个地区具有不同流行特征的ＲＳＶ地方株（Ａ亚型）５株，进行ＲＳＶＦ蛋白全基因的核苷酸序列分析。方法以提取的病毒ｍＲＮＡ为模板进行ＲＴ－ＰＣＲ扩增、目的基因的克隆及序列测定，对地方株及原型株的序列进行比较分析。结果地方株Ｆ蛋白基因与原型株Ａ２株有很高的同源性，核苷酸全序列的同源性为９５．１％～９６．１％，氨基酸同源性为９６．７％～９７．４％。核苷酸有义突变率为２２．６％～２５．９％。３非编码区的核苷酸序列比蛋白编码区变异显著。河北地方株（Ｅ７３株）在３非编码区有６个核苷酸的插入。Ｆ２亚单位的氨基酸变异高于Ｆ１亚单位。在北京地方株（ＺＨＳ１３株）Ｆ１亚单位内，由具有中和能力单克隆抗体所识别的抗原表位区中存在一个氨基酸的变异。结论我国ＲＳＶ地方株与原型株之间的Ｆ蛋白基因尽管存在一定的变异，但仍有很高的同源性。地方株间Ｆ蛋白的核苷酸、氨基酸变异的位置及形式很相似，提示我国ＲＳＶ的不同流行特征可能并非由于Ｆ蛋白的基因变异所致。     

Chromosomal mapping of the uracil permease gene of Saccharomyces cerevisiae

Summary The gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome. In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped. After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.     

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae

Summary Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T₂ gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T₂ revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T₂ activity than wild-type strains.     

Cloning and sequence analysis of the N gene of porcine epidemic diarrhea virus LJB/03

The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) strain LJB/03 which was previously isolated in Heilongjiang province, China, was cloned, sequenced and compared with published sequences of other avian and mammalian coronavirus. The nucleotide sequence encoding the entire N gene open reading frame (ORF) of LJB/03 was 1326 bases long and encoded a protein of 441 amino acids with predicted Mr of 49 kDa. It consisted of 405 adenines (30.5%), 294 cytosines (22.1%), 329 guanines (24.8%) and 298 thymines (22.5%) residues. Sequence comparison with other PEDV strains selected from GenBank revealed that the LJB/03 N gene has a high sequence homology to those of other PEDV isolates, 97.4% with JS2004, 95.6% with chinju99, 96.6% with Br1/87, and 96.8% with CV777. The encoded protein shared 96.4% amino acid identities compared with CV777, 96.1% with Brl/87, 98% with JS2004, 96.90% with chinju99, respectively. The amino acid sequence contained seven potential protein kinase C phosphorylation sites, nine Casein kinase II phosphorylation sites, one Tyrosine kinase phosphorylation site, two cAMP- and cGMP-dependent protein kinase phosphorylation sites.     

The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene

Summary As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to lie in its single G,C rich cluster, which differs in just one nucleotide from the standard ori ^s sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergencies (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.     

Cloning and nucleotide sequence of the gene encoding arginine deiminase of Mycoplasma arginini.

The existence of a mycoplasmal arginine deiminase which catalyzes the conversion of L-arginine to L-citrulline has been postulated. Here we show the partial amino acid sequence of arginine deiminase of Mycoplasma arginini and the complete nucleotide sequence of the arginine deiminase gene of M. arginini. The open reading frame deduced from this sequence consists of 1,230 bp encoding 410 amino acids. The mature form of this enzyme contains 409 amino acids after the deletion of the first methionine. In this open reading frame, TGA nonsense codons are used as tryptophan codons; this usage was verified by determination of the amino acid sequence. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 46,372. Recently, the nucleotide sequence of the arginine deiminase gene of M. arginini was reported by Kondo et al. (K. Kondo, H. Sone, H. Yoshida, T. Toida, K. Kanatani, Y.-M. Hong N. Nishino, and J. Tanaka, Mol. Gen. Genet. 221:81-86, 1990). However, their sequence differed from ours in several places and especially at the C terminus.     

L-ornithine transaminase synthesis in Saccharomyces cerevisiae: Induction by allophanate,intermediate and inducer of the urea degradative pathway adds to arginine induction

Summary Yeast ornithine transaminase is known to be induced by arginine and ornithine, through the action of regulatory elements common to arginase induction. We show here that it is subject to a second induction circuit, that which is responsible for urea amidolyase and urea permease induction by allophanate and defined by the regulatory mutants durL ^– and durM ^–     

Complete nucleotide sequences and genome organization of four chimpanzee adenoviruses

The complete nucleotide sequences for four adenoviruses-Simian Adenoviruses 21, 22, 23, and 24, originally isolated from chimpanzees, were determined. The genome organization of the chimpanzee adenoviruses was found to be similar to that of other adenoviruses. The viral gene products of the adenoviruses Simian Adenoviruses 22, 23, and 24 are very closely related to those of the (previously sequenced) chimpanzee adenovirus Simian Adenovirus 25. Simian Adenovirus 21 is most similar to human subgroup B adenoviruses HAdV-3, HAdV-7, and HAdV-35. Analysis of the capsid proteins hexon and fiber of the chimpanzee adenoviruses also supports the placement of Simian Adenovirus 21 in subgroup B and Simian Adenoviruses 22, 23, and 24 in subgroup E.     

A nucleotide sequence involved in replicative transformation of a filamentous fungus

Replicative plasmids generated through in-vivo recombination have been identified among transformants of the fungus Pleurotus ostreatus. In addition to sequences from a standard selection vector (pAN7-1), these recombinant plasmids contain recombined sequences of chromosomal origin conferring replicative potential upon the vector. One such recombined sequence, an 1148-bp insert into plasmid pP01, has been characterized. This sequence has been analyzed for secondary structural features as well as for consensus sites affiliated with origins of replication (ori) in other eukaryotic systems. The 1148-bp insert lacks an ORF and does not contain an acceptable match to the commonly identified 11-bp ars consensus sequence (A/TTTTATA/GTTTA/T) for autonomous replication in the yeast Saccharomyces cerevisiae. The analysis, however, revealed a cluster of three hairpin-loop-forming subsequences with individual G_25°C free energy values of-7.6,-6.4 and-5.2 kcal mol^-1. Also found were two 7-bp analogues to centromere-affiliated sequences recognized in other fungi, as well as several putative gyrase recognition sites comparable to the 9-bp S. cerevisiae/E. coli gyrase-binding consensus sequence. Sequences comparable to the ori of the yeast 2-m plasmid or to various sequences associated with ori of yeast/fungal mitochondrial DNAs (mtDNA) were not present in the 1148-bp insert. Replication of pP01 appears rather to involve a replication of chromosomal derivation devoid of an ars-type consensus.     

献血员中TT病毒DNA检测及部分基因序列分析

目的探讨献血员中一种与输血后肝炎有关的病毒—ＴＴＶ的感染情况。方法采用ＴＴＶ基因组ＯＲＦ１区的套式聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）方法检测２６２份献血员血清标本。结果在血清丙氨酸转氨酶（ＡＬＴ）异常、ＨＢｓＡｇ及抗－ＨＣＶ阴性的５８份献血员血清标本中，１８份ＴＴＶＤＮＡ阳性，ＴＴＶＤＮＡ的阳性检出率为３１０％；在２０４份正常献血员血清标本中检出３０份（１４７％）ＴＴＶＤＮＡ阳性，ＴＴＶＤＮＡ的阳性检出率明显低于ＡＬＴ异常献血员人群（χ２＝３８１，Ｐ＜００５）。序列分析结果显示，其序列与日本株和中国株相应位置核苷酸序列的同源性大于９７％，可能属同一基因型。结论提示我国献血员中存在ＴＴＶ感染，ＴＴＶ存在“健康携带状态”，输血可能成为传播ＴＴＶ感染的途径之一     

Organization and nucleotide sequence of the broad bean chloroplast genes trnL-UAG,ndhF and two unidentified open reading frames

Summary We have determined the nucleotide sequence of a 6.9 kbp BamHI-Xbal fragment of broad bean chloroplasts. Part of this fragment (subfragment BglII-CIal) is known to contain three tRNA genes (trnL-CAA, trnlrUAA and trnF). We have now further identified a gene coding for the third tRNA^Leu isoacceptor (trnL-UAG) which is located close to trnF. The BamHI-Xbal fragment also contains the gene for subunit 5 of NADH dehydrogenase (ndhF) and two unidentified open reading frames (ORFx and ORF48). ORFx shares a high sequence homology with the long reading frames of tobacco (ORF1708), spinach (ORF2131), and liverwort (ORF2136), while ORF48 shares sequence homology with ORF69 of liverwort and ORF55 of tobacco.     

Yeast ts secretory mutation rgs1 is suppressed by the SEC4 gene of Saccharomyces cerevisiae

Yeast rgs1 cells accumulate secretory vesicles in the cytoplasm and stop the secretion of proteins at the restrictive temperature. The ts mutation rgs1 may be suppressed by several different genes; the S. cerevisiae SEC4 gene, encoding the small G-protein involved in the late secretory stage, is one of them. Synthetic lethality of the double rgs1 sec4 mutant is demonstrated.     

Complete nucleotide sequence of the influenza C/California/78 virus nucleoprotein gene

The complete nucleotide sequence of RNA segment 5 of the influenza C/California/78 (C/Cal/78) virus was determined by using cloned cDNA derived from viral RNA. The gene contains 1809 nucleotides and can code for a protein of 565 amino acids with a molecular weight of 63 525. The RNA 5 protein of the influenza C/Cal/78 virus possesses two short regions which share a high degree (60–83%) of sequence homology with the nucleoproteins of influenza A and B viruses. These and other structural features of the RNA 5 protein suggest that RNA 5 of influenza C viruses codes for the nucleoprotein. The data also suggest that influenza C viruses are orthomyxoviruses, but that they are more distantly related to either type A or type B viruses than are influenza A and B viruses to each other.