Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation

Summary The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than I I min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.     

Functional interactions between the gene tetanic and the Shaker gene complex of Drosophila

Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII [l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K⁺ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.     

Escherichia coli parA is an allele of the gyrB gene

Summary A thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations. The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd. A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110. The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA. This mutation was linked with the Par phenotype as shown unambiguously by 4,6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background. Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene. This strongly suggests that parA is an allele of gyrB.     

Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination

A recN ^– (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF recH and addAB genes, when present in an otherwise Rec⁺ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10²- to 10⁴-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for, DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.     

单碱基编辑系统介导欧拉藏绵羊GDF9、FecB多胎基因的定点突变

本研究旨在利用单碱基编辑系统(single base editing system)实现欧拉藏绵羊成纤维细胞FecB和GDF9基因靶位点A到G和C到T的碱基替换并检测其编辑效率。首先设计合成靶向欧拉藏绵羊FecB和GDF9基因的sgRNA序列,再分别连接至epi-ABEmax、epi-BE4max质粒,构建载体并电转至欧拉藏绵羊成纤维细胞,最后对阳性细胞FecB和GDF9基因进行Sanger测序鉴定靶位点突变结果,并通过T-A克隆估算单碱基编辑系统的编辑效率。结果显示获得了靶向欧拉藏绵羊FecB和GDF9基因的sgRNA,并构建使欧拉藏绵羊FecB和GDF9基因单碱基突变的载体,FecB基因靶位点编辑效率为39.13%,GDF9基因靶位点(G260、G721、G1184)编辑效率分别为10.52%、26.67%和8.00%。本研究运用单碱基编辑系统在欧拉藏绵羊成纤维细胞上实现了FecB和GDF9基因靶位点突变,为改良欧拉藏绵羊一胎多羔的繁殖性状奠定理论基础。     

拟南芥bso-1突变体的基因定位及表型分析

通过EMS化学诱变在拟南芥Columbia(Col-0)野生型突变体库中筛选获得1株器官显著增大的突变体,命名为big size organ1(bso-1)。遗传分析表明,bso-1受单个隐性核基因控制。表型观察发现,突变体植株的幼苗、花、果荚及种子与野生型相比都表现出明显的增大。组织切片结果显示,突变体种子的增大主要由胚细胞个体增大导致胚体积增大而实现,因此突变体种子的重量也较野生型有明显增加。利用图位克隆方法将相关基因初步定位在4号染色体上SSLP标记T5L19与F28M11之间58kb区间内,生物信息学分析显示此区间内未见调控植物器官大小发育相关的已知基因的报道。该研究结果为进一步克隆bso-1突变体相关基因及探讨其在控制植物器官发育尤其是种子发育过程中的作用奠定了基础。     

Genetic structure and domains of DNA polymerase III of Bacillus subtilis

Summary We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus' of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3 to 5 exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.Abbreviations HPUra p-hydroxyphenyl azouracil - nt nucleotide - PCR polymerase chain reaction     

Characterization of the cyrl-2 UGA mutation in Saccharomyces cerevisiae

Summary cyrl-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyrl-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyrl-2 mutation. Construction of chimeric genes restricted the region containing the cyrl-2 mutation, and the cyrl-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyrl-2 mutation site result in null mutations. The complete open reading frame of the cyrl-2 gene expressed under the control of the GAL1 promoter complemented cyrl-dl in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyrl-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.     

Two new mutations of the human BCHE gene (IVS3-14T>C and L574fsX576)

The genetic variation of human butyrylcholinesterase is associated with the majority of prolonged cases of apnea in patients submitted to the muscle relaxant succinylcholine. The present study reports two new mutations of the BCHE gene in 346 Euro-Brazilians: IVS3-14T>C found in five heterozygotes (allele frequency: 0.72 ± 0.32%) and L574fsX576 found in one heterozygote (allele frequency: 0.14 ± 0.14%). These two variants were not found in 85 Guarani Amerindians. It is not expected that the IVS3-14T>C mutation may interfere in the splicing process and that the mutation found in exon 4 (L574fsX576) may disturb BChE tetramerization and activity.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.     

TFS1: A suppressor of cdc25 mutations in Saccharomyces cerevisiae

Summary The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5 flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5 regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。