Recombination in Relation to Ultraviolet Sensitivity in CHLAMYDOMONAS REINHARDI

A mutant strain of Chlamydomonas reinhardi which is UV sensitive as a result of a single-gene chromosomal mutation has also been found to exhibit reduced recombination. In crosses homozygous for the mutant allele, a reduction in recombination frequency was demonstrated in two different linkage groups and in three different genetic backgrounds. Thus a single mutation affecting UV sensitivity also has a possible effect on recombination. Such a mutant could be analogous to the rec(-) mutants discovered in E. coli and as such be useful in the study of recombination mechanisms. Three additional UV-sensitive isolates were tested. Recombination was not altered in these mutant strains.     

Two new genes (smhA and lytE) apparently functionally related to the murH gene of Escherichia coli

The murH mutant of Escherichia coli exhibits temperature-sensitive growth and lysis at the restrictive temperature. Temperature-resistant derivatives of the mutant occurred at a frequency of about 3 X 10(-6). All of the seven independent isolates examined were shown to be pseudorevertants carrying extragenic suppressors of murH, which mapped at 24.5 min on the linkage map. One allele, apparently representing a new locus, designated smhA, was characterized further. The smhA mutation by itself conferred no recognizable phenotype. However, smhA suppressed the temperature-sensitive lysis phenotype of the murH mutant. The smhA mutant acquired a spontaneous mutation in another new gene, designated lytE, which was mapped at 25 min. The lytE mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. The lytE mutation was suppressed by smhA as well as by another suppressor of murH designated smhB. The suppressor activity of smhA was apparently relatively specific in that smhA failed to prevent lysis caused by either mutational or antibiotic-induced blocks in peptidoglycan synthesis. The possibility that the smhA and lytE genes are functionally related to murH is considered.     

The use of mitochondrial mutants in the isolation of hybrids involving industrial yeast strains

Summary Methods for the isolation of hybrids in which one or both of the parental strains are industrial yeasts, using mitochondrial mutations as markers for the selection and isolation of the hybrids, are described. The systems used included crosses of industrial strains with auxotrophic laboratory strains which also carried a mitochondrial antibiotic resistance mutation, crosses using an auxotrophic laboratory strain and a petite mutant of an industrial strain carrying a rescuable antibiotic resistance mutation, and crosses using a petite mutant of an industrial strain, carrying a rescuable mitochondrial mutation for antibiotic resistance and a respiratory-competent industrial strain which carried some other marker.     

Analysis of Dysgenesis-Induced Lethal Mutations on the X Chromosome of a Q Strain of DROSOPHILA MELANOGASTER

The Q strain known as v6 was tested for its ability to induce X-linked lethal mutations in male and female hybrids from crosses with M strains in the P-M system of hybrid dysgenesis. All measurements of the mutation rate were made on the X chromosome derived from the v6 strain. The lethal rate for young hybrid males from the cross M female X v6 male was 1.11% per chromosome. For older males, it was only 0.44%, suggesting that there is less mutational or more repair activity in the germ cells of the older males or that mutant cells are selectively eliminated as the hybrid males age. The lethal rate for hybrid females from comparable crosses was approximately the same for both ages that were tested. However, it was substantially less than the rate for the hybrid males--only 0.26% per chromosome. Genetically identical hybrid females from reciprocal crosses also showed a low mutation rate, 0.13% per chromosome. Again, there was no difference between young and old flies. Mapping experiments established that most of the lethal mutations that were recovered from the male and female hybrids were located in two regions on the X chromosome, one between bands 14B13 and 15A9 , the other between bands 19A1 and 20A , which encompasses the maroonlike locus. More refined mapping of the lethals in the maroonlike region demonstrated that the vast majority of these affected a single gene located in band 19C4 . Cytological analysis of the lethal chromosomes revealed that several carried rearrangements, including inversions, duplications and deficiencies. Chromosome breakage occurred primarily in bands 14D1 -3 and 18F- 20A , and most of the breaks in the latter segment were located in 19C . However, rearrangements involving 19C and mutations of the gene in 19C4 were mutually exclusive events. In situ hybridization of a P element probe to the chromosomes of v6 demonstrated that P elements reside at a minimum of five sites on the X chromosome. These P element sites correspond to the mutational and breakage hot spots on that chromosome. The combined genetic and cytological data imply that most of the X-linked lethal mutations that occur in M X v6 hybrids are due to local P element action. Consideration of these and other data suggest that v6 is a weak P strain in the P-M system of hybrid dysgenesis and that other Q strains might also be regarded in this way.     

Behavior of hobo and P transposons in yellow2-717 unstable line of Drosophila melanogaster and its derivatives after crossing with a laboratory strain

Using fluorescent in situ hybridization technique (FISH), the frequency of hobo and P mobile elements transpositions on X chromosomes from the y2-717, isolated from the Uman' population of Drosophila melanogaster, as well as from its phenotypically normal and mutant derivatives, obtained as a result of crosses the males examined with the C(I)DX, ywf/Y females, was evaluated. It was demonstrated that the maximum frequency of hobo transpositions on X chromosomes of the males from derivative strains, subjected to repeated hobo-dysgenic crosses reached a value of 1.2 x 10(-2) per site per X chromosome per generation. The number of hobo copies in male X chromosomes from derivative strains was 3 times higher than in the original initial strain. Furthermore, the "old" hobo sites remained unchanged. In derivative strains, the frequency of hobo insertions was higher than that of excisions. One of the derivative strains, y1t-717alk3-2, was characterized by high intra-strain instability of hobo element localization. In the y2-717a1k3 and y1t-717alk3-2 strains a large inversion, In(1)1B; 13CD, was described. At the absence of the full-sized P element in the strains involved in crosses, maximum frequency of P element transpositions in the derivative strains reached a value of 1.2 x 10(-2) per site per X chromosome per generation.     

Detection and characterization of plasmids in Pseudomonas glycinea.

Pathogenic strains of Pseudomonas glycinea were shown to possess plasmid deoxyribonucleic acid by dye-buoyant density gradient centrifugation. The size and number of plasmids of four different isolates were determined by neutral sucrose gradient centrifugation. Two isolates were found to harbor a single plasmid; however, they differed in size, having molecular weights of 43 X 10(6) and 54 X 10(6). Two other isolates each contained two different plasmids. Plasmids with molecular weights of 43 X 10(6) and 73 X 10(6) were observed in one isolate, and the other carried plasmids with molecular weights of 25 X 10(6) and 87 X 10(6). An auxotrophic mutant derived from the latter strain was found to contain plasmids of identical size. The plasmids were found to be under stringent control of replication, having plasmid copies of 1.0 to 2.7 per chromosome equivalent. By the dye-cesium chloride technique, the mutant showed twice as much covalently closed circular deoxyribonucleic acid as did the parental strain.     

Repeat-induced point mutation (RIP) in crosses with wild-isolated strains of Neurospora crassa: evidence for dominant reduction of RIP

Seventy-one wild-isolated strains of Neurospora crassa were examined for their ability to support repeat-induced point mutation (RIP) in the erg-3 locus. RIP was exceptionally inefficient but detectable in crosses with the strain FGSC 430 from Adiopodoume, Ivory Coast. We could find no consistent differences in ascospore yields when wild isolates identified as "low-RIP" or "high-RIP" strains were crossed with strains bearing the segmental duplication Dp(IIIR > [I; II])AR17. This suggested that RIP may not be responsible for the barren phenotype of crosses involving segmental duplication strains.     

普通小麦远缘杂交F1代表现型研究

小麦远缘杂交试验采用常规杂交方法,不进行胚培养,在自然条件下,实现了普通小麦与小黑麦(6X,AABBRR)、柱穗山羊草(2X,DD)、人工合成六倍体小麦(6X,AABBDD)、CIMMYT人工合成小麦(6X,AABBDD)的杂交.发现在F1代中,出现了2～7种植株表现型、7种性状分离现象;小麦颖壳颜色变化可能受多个加性基因控制;杂交F2代结实率较F1代有数倍或十余倍的增长.     

Altered Mating-Type Identity in the Fungus Podospora Anserina Leads to Selfish Nuclei, Uniparental Progeny, and Haploid Meiosis

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strains, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strains carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant X wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.     

Dominant cataract and recessive specific locus mutations in offspring of X-irradiated male mice

Male mice were X-irradiated with 3.0 + 3.0 Gy or 5.1 + 5.1 Gy (fractionation interval 24 h). The offspring were screened for dominant cataract and recessive specific locus mutations. In the 3.0 + 3.0-Gy spermatogonial treatment group, 3 dominant cataract mutations were confirmed in 15 551 offspring examined and 29 specific locus mutations were recovered in 18 139 offspring. In the post-spermatogonial treatment group, 1 dominant cataract mutation was obtained in 1120 offspring and 1 recessive specific locus mutation was recovered in 1127 offspring. The induced mutation rate per locus, per gamete, per Gy calculated for recessive specific locus mutations is 2.0 X 10(-5) in post-spermatogonial stages and 3.7 X 10(-5) in spermatogonia. For dominant cataract mutations, assuming 30 loci, the induced mutation rate is 5.0 X 10(-6) in the post-spermatogonial stages and 1.1 X 10(-6) in spermatogonia. In the 5.1 + 5.1-Gy spermatogonial treatment group, 3 dominant cataract mutations were obtained in 11 205 offspring, whereas in 13 201 offspring 27 recessive specific locus mutations were detected in the spermatogonial group. In the post-spermatogonial treatment group no dominant cataract mutation was observed in 425 offspring and 2 recessive specific locus mutations were detected in 445 offspring. The induced mutation rate per locus, gamete and Gy in spermatogonia for recessive specific locus mutations is 2.8 X 10(-5) and for dominant cataract mutations 0.9 X 10(-6). In post-spermatogonial stages, the mutation rate for recessive specific locus alleles is 6.2 X 10(-5). In the concurrent untreated control group, in 11 036 offspring no dominant cataract mutation and in 23 518 offspring no recessive specific locus mutation was observed. Litter size and the number of carriers at weaning have been determined in the confirmation crosses of the obtained dominant cataract mutants as indicators of viability and penetrance effects. Two mutants had a statistically significantly reduced litter size and one mutant had a statistically significantly reduced penetrance.     

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae

Glutathione-deficient (gsh-) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh-:GSH+ phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh- mutants belong to one complementation group.     

Identification and characterization of Schizosaccharomyces pombe asp1(+), a gene that interacts with mutations in the Arp2/3 complex and actin.

The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1(+). The asp1(+) open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1(+) nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1(+) leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Delta strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.     

Effect of auxotrophic starvation on mitochondrial marker transmission in the cdc8 mutant of Saccharomyces cerevisiae

Summary Crosses were made using strains of S. cerevisiae which carried mitochondrial markers conferring resistance to erythromycin and chloramphenicol. The effect of auxotrophic starvation of one parent prior to mating on the transmission of its mitochondrial markers was studied in different crosses relative to the presence of the cdc8 nuclear mutation (a temperature-sensitive DNA replication).In crosses between two cdc8 mutant strains, auxotrophic starvation of one of the haploid parental strains prior to mating caused a marked decrease of its mitochondrial marker transmission to the diploid progeny of the cross. The transmission decreased as a function of the time of starvation. This effect was not observed in the cross between two wild type strains and in crosses of starved cdc8 phenotypic revertants with cdc8 mutant strains. Only a small, if any, effect of starvation on mitochonrial marker transmission was observed when starved cdc8 mutant strains were crossed either with their phenotypic revettants or with the wild-type strains.In one of the haploid parental strains the starvation increased the frequency of petites as a function of starvation time, while in the other this effect was not observed.In the progeny of cdc8xcdc8 crosses (both in starvation experiments and in control crosses) an increased frequency of diploid petite cells accompanied by a decreased frequency of recombination between mitochondrial markers was noticed.The influence of the cdc8 mutation on the transmission of mitochondrial markers is discussed in terms of high frequency of ^– molecule formation in cdc8 strains.     

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis.

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.     

Mapping of the gene specifying aminoglycoside 3''-phosphotransferase II on the Pseudomonas aeruginosa chromosome.

We examined the aminoglycoside inactivation enzymes in Pseudomonas aeruginosa strains, seven clinical isolates and seven laboratory strains without plasmids. All strains were found to possess the enzyme aminoglycoside 3'-phosphotransferase II [APH(3')-II]. We isolated an APH(3')-II-deficient mutant from a PAO strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. By plasmid (FP5 or R68.45)-mediated conjugation, we determined the locus of the gene specifying the APH(3')-II between trp-6 and pro-82 on the PAO chromosome and designated this gene aphA. It was concluded that the intrinsic resistance of P. aeruginosa to kanamycins, neomycins, paromomycins, ribostamycin, and butirosins was due to this newly determined gene.     

Asparaginase II of Saccharomyces cerevisiae: selection of four mutations that cause derepressed enzyme synthesis.

A positive selection method was used to isolate four Saccharomyces cerevisiae mutations that cause derepressed synthesis of asparaginase II. The four mutations (and1, and2, and3, and4) were neither closely linked to each other nor linked to previously characterized mutations (asp3, asp6) which cause the complete loss of asparaginase II activity. One of the new mutations (and4) was shown to be allelic to gdh-CR, a pleiotropic mutation which causes derepressed synthesis of a number of enzymes of nitrogen catabolism.     

Molecular Characterization of Invasive Meningococcal Isolates from Countries in the African Meningitis Belt before Introduction of a Serogroup A Conjugate Vaccine

Background

The serogroup A conjugate meningococcal vaccine, MenAfriVac, was introduced in mass vaccination campaigns in December 2010 in Burkina Faso, Mali and Niger. In the coming years, vaccination will be extended to other African countries at risk of epidemics. To document the molecular characteristics of disease-causing meningococcal strains circulating in the meningitis belt of Africa before vaccine introduction, the World Health Organization Collaborating Centers on Meningococci in Europe and United States established a common strain collection of 773 isolates from cases of invasive meningococcal disease collected between 2004 and 2010 from 13 sub-Saharan countries.

Methodology

All isolates were characterized by multilocus sequence typing, and 487 (62%) were also analyzed for genetic variation in the surface antigens PorA and FetA. Antibiotic susceptibility was tested for part of the collection.

Principal Findings

Only 19 sequence types (STs) belonging to 6 clonal complexes were revealed. ST-5 clonal complex dominated with 578 (74.8%) isolates. All ST-5 complex isolates were remarkably homogeneous in their PorA (P1.20,9) and FetA (F3-1) and characterized the serogroup A strains which have been responsible for most epidemics during this time period. Sixty-eight (8.8%) of the 773 isolates belonged to the ST-11 clonal complex which was mainly represented by serogroup W135, while an additional 38 (4.9%) W135 isolates belonged to the ST-175 complex. Forty-eight (6.2%) serogroup X isolates from West Africa belonged to the ST-181 complex, while serogroup X cases in Kenya and Uganda were caused by an unrelated clone, ST-5403. Serogroup X, ST-181, emerged in Burkina Faso before vaccine introduction.

Conclusions

In the seven years preceding introduction of a new serogroup A conjugate vaccine, serogroup A of the ST-5 clonal complex was identified as the predominant disease-causing strain.     

中国耐多药结核分枝杆菌临床分离株rpoB基因突变特点

为阐明中国耐多药结核分枝杆菌临床分离株rpoB基因的突变特征,对86株结核分枝杆菌临床分离株rpoB基因两个区域,包括81个碱基利福平抗药性决定区(rifampin resistance determining region,RRDR)和V176F区进行序列测定。其中72株耐多药分离株中的65株rpoB基因的RRDR区存在22种不同类型突变、21种点突变和一个插入突变。最常见的突变部位分别位于密码子531(41％)、526(40％)和516(4％),10％耐药分离株未检测到突变。鉴定了RRDR内6个新的等位基因,以及RRDR外部区域5个新的突变。所有分离菌株V176均无突变。     

Isolation and Characterization of a Recombination-Deficient Hfr Strain

The isolation of a rec(-) Hfr strain of Escherichia coli K-12 is described. The method used consisted of mating AB2463 F(-) Rec(-) His(-) Lac(-) with P4X6 Hfr Rec(+) His(+) Lac(+), selecting Rec(-) His(-) Lac(+) recombinants, and searching for Hfr strains. One Hfr rec(-) strain, no. 12, was used as donor in crosses with Rec(+) and Rec(-) recipients. Crosses with Rec(+) recipients are fertile, and those with Rec(-) recipients are almost infertile, the frequency of recombinants being 10(-2) to 10(-3) that found with Rec(+) recipients. The Rec(-) mutant marker is transfered to and integrated into Rec(+) recipients. Zygotic induction of prophage lambda is observed in crosses between two Rec(-) strains. In crosses of F(-) Rec(-) with Hfr Rec(-), the gradient of integration frequencies for markers progressively more distant from the origin is steeper than in the Rec(+) x Rec(-) or the Rec(-) x Rec(+) crosses.     

肺炎链球菌pbp2b和pbp1a基因突变与青霉素耐药的相关性研究

目的探讨耐青霉素肺炎链球菌pbp2b和pbp1 a基因的突变与青霉素耐药的关系,为明了肺炎链球菌的耐药性变异机制,防治其感染提供实验依据。方法从呼吸道感染患儿痰标本中分离肺炎链球菌163株,液体培养基连续稀释法测定其对青霉素的最小抑菌浓度(M IC),套式聚合酶链反应(nPCR)扩增pbp2b和pbp1 a基因,扩增产物直接DNA测序,所测序列与青霉素敏感株(SPN R6)的基因序列进行比较,并分析其氨基酸结构的改变。结果 163株肺炎链球菌中检出青霉素敏感菌75株,中度敏感17株,青霉素耐药菌71株(44%)。耐药菌中58株存在pbp2b突变(81.7%),其中,56株为点突变,2株为CCT插入突变;在27株有pbp2b基因突变的B型和C型耐药菌中,21株出现了不同程度的pbp1 a基因突变。PBP2B氨基酸结构改变以苏氨酸变为丙氨酸、精氨酸变为赖氨酸为主,PBP1A以丙氨酸变为苏氨酸、谷氨酸变为天门冬氨酸为主。结论肺炎链球菌的pbp2b和pbp1 a基因突变与对青霉素的耐药性密切相关,PBP2b突变导致低水平耐药;PBP2b和PBP1A突变导致高水平耐药。