Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene.     

抗真菌肽CGA-N46基因多顺反子表达载体的构建与鉴定

目的:为提高抗真菌肽CGA-N46表达量,对该基因多顺反子表达进行研究.方法:以pEASY-Blunt为克隆栽体,以“pET-30a rbs序列-起始密码子-CGA-N46编码序列-终止密码子”为外源片段,利用同尾酶Nhe I、Spe I和Xba I,构建了含有上述外源片段1、3、5、8拷贝的重组载体pT-CAN46、...     

The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA.

The use gene of Salmonella typhimurium was previously identified by a mutation conferring sensitivity to uracil in glucose minimal medium. The use gene was cloned and identified as an allele of argU encoding a tRNA for a minor arginine codon (CGG). The uracil-sensitive phenotype was shown to result from a base substitution in the anticodon stem of this tRNA.     

一个46,XY"女性"不育症家系的遗传学分析

运用常规的染色体G带分析和基因分析技术对-46,XY男性女性化家系进行遗传学分析,发现：先证者及其妹妹的染色体核型为46,XY,其母亲和父亲的核型正常;对睾丸决定基因（SRY）和雄激素受体基因（AR）进行突变检测,在SRY基因的整个编码区中没有发现突变,而AR基因的第7个外显子的第840个密码子由CGT（编码精氨酸）变为CAT（编码组氨酸）,这一改变可能是导致核型为46,XY女性化而发生不育。     

Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.     

Genetic Analysis of a Family with 46, XY “Female” Associated with Infertility

Genetic analysis of a family with 46, XY “female” associated with infertility was performed using the conventional G-banding and gene mutation screening. The karyotypes of the two female patients were 46, XY and those of their parents were normal. The mutation screenings in sex-determining region Y (SRY) gene and androgen receptor (AR) gene were carried out. No mutation has been found in the whole coding sequence of SRY gene. The mutation of codon 840 CGT (arginine) to CAT (histidine) of AR gene led to the infertility in the patients. The patients showed androgen insensitivity. The clinical phenotype of theirs presented more deleteriously than and different from the one reported before, though they had the same mutation of codon 840 CGT (arginine) to CAT (histidine) of AR gene, which was very different from the mutation of 840 CGT (arginine) to TGT (cysteine) at the same codon.     

Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin-like antibiotics

The nucleotide sequence of a 1883 bp fragment isolated from a resistance plasmid harbored by a Staphylococcus aureus clinical isolate and carrying the gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin family has been determined. The sequence contains one open reading frame which extends from the ATG codon at nt 641 to a TGA codon at nt 1537 and which potentially codes for a protein of 33.035 Da. This value is in agreement with the apparent size (33 kDa) of the protein observed, in minicell extracts. Inactivation of the B components of the virginiamycin antibiotics as well as resistance to these antibiotics were expressed in a virginiamycin sensitive mutant of Escherichia coli recipient containing the gene on a high copy number plasmid.     

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci.     

Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.     

Albinism in the American mink (Neovison vison) is associated with a tyrosinase nonsense mutation

Albino phenotypes are documented in various species including the American mink. In other species the albino phenotypes are associated with tyrosinase (TYR) gene mutations; therefore TYR was considered the candidate gene for albinism in mink. Four microsatellite markers were chosen in the predicted region of the TYR gene. Genotypes at the markers Mvi6025 and Mvi6034 were found to be associated with the albino phenotype within an extended half-sib family. A BAC clone containing Mvi6034 was mapped to chromosome 7q1.1-q1.3 by fluorescent in situ hybridization. Subsequent analysis of genomic TYR sequences from wild-type and albino mink samples identified a nonsense mutation in exon 1, which converts a TGT codon encoding cysteine to a TGA stop codon (c.138T>A, p.C46X; EU627590). The mutation truncates more than 90% of the normal gene product including the putative catalytic domains. The results indicate that the nonsense mutation is responsible for the albino phenotype in the American mink.     

人α-心钠素全基因的化学合成

用固相亚磷酰胺法(solid-phase phosphoramidite method)合成了人α-心钠素(简称α-hANP)的全基因。合成的α-hANP基因全长为96bp,包括编码α-hANP的结构基因、基因5＇端的谷氨酸(作为表达产物用内肽酶Glu-C专一裂解的位点)密码子GAA、基因3＇端的终止信号TGA及基因两端的接头部分。基因分7个寡聚核苷酸片段在DNA合成仪上合成,然后一次性酶促连接成为完整的α—hANP双链基因。化学合成的基因克隆到M13载体上,经分子杂交鉴定筛选出的α-hANP基因克隆株,用双脱氧链终止法进行序列分析,证明核苷酸顺序正确。     

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.     

Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates

In this study, we compared the potential of amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of tandem repeats (VNTR) of the P97 encoding gene, as possible methods for typing an international collection of Mycoplasma hyopneumoniae isolates. All techniques showed a typeability of 100% and high intraspecific diversity. However, the discriminatory power of the different techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene (>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97 (<0.92). Other, preferentially well spread, tandem repeat regions should be included in order for this latter technique to become valuable for typing purposes. RAPD was also found to be a less interesting typing technique because of its low reproducibility between different runs. Nevertheless, all molecular techniques showed overall more resemblance between strains isolated from different pigs from the same herd. On the other hand, none of the techniques was able to show a clear relationship between the country of origin and the fingerprints obtained. We conclude that AFLP and an earlier described PFGE technique are highly reliable and discriminatory typing techniques for outlining the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an equally useful alternative for demonstrating intraspecific variability, although the generation of sequence variability of the gene remains unclear and must be further examined.     

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline)----CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 his G gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.     

Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids

Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39 degrees C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.     

CL-46, a novel collectin highly expressed in bovine thymus and liver

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via alpha-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the alpha-helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans approximately 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.     

Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product.

The formate dehydrogenase (FDHF) of Escherichia coli is a selenocysteine-containing protein that occurs as a component of the formate-hydrogen lyase complex. The gene encoding this 80 kd polypeptide contains a TGA codon in the open reading frame. Several indirect lines of evidence showed earlier that the selenocysteine residue in the protein is inserted co-translationally in a TGA (UGA) dependent process. Direct proof that the selenocysteine is present in the polypeptide in the position corresponding to TGA as predicted from the gene sequence was obtained by automated amino acid sequence analysis of a 75Se-containing peptide isolated from the protein. Construction of a fusion gene comprising a small segment of the fdhF gene linked to the lacZ gene as reporter greatly facilitated isolation of the selenocysteine-containing protein. Subsequent cleavage of this isolated gene product with endoproteinase Asp-N gave rise to an easily purified small selenocysteine-containing peptide that was amenable to amino acid sequence analysis.