Local Transposition of P Elements in Drosophila Melanogaster and Recombination between Duplicated Elements Using a Site-Specific Recombinase

The transposase source Δ2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic while gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.     

The Bxb1 Recombinase Mediates Site-Specific Deletion in Transgenic Wheat

The utility and commercial potential of genetically engineered (GE) plants would benefit from the use of site-specific recombination systems that enable efficient excision of the marker genes used to identify transformants. Although wheat is one of the most important food crops in the world, GE varieties have yet to be put into commercial production. To develop the Bxb1 recombination system (derived from the Mycobacterium smegmati bacteriophage Bxb1) for site-specific marker gene removal in transgenic wheat, we used biolistics to introduce into the wheat genome a codon optimized Bxb1 recombinase gene (BxbNom) under the control of the maize ubiquitin-1 promoter (Ubi1). Recombinase activity was monitored using a GUSPlus reporter gene activation assay. BxbNom recombinase-mediated excision of an att site-flanked stuffer DNA fragment activated ??-glucuronidase reporter activity in callus, endosperm, and leaves in transient assays. The system also detected activity in leaves and endosperm of progeny of multiple independent transgenic wheat lines stably expressing BxbNom. Our results demonstrate that the Bxb1 recombinase is heritable in transgenic wheat plants and performs site-specific excision, providing a useful tool for generating marker-free GE plants. Establishment of wheat lines capable of efficiently excising unneeded marker genes removes one potential barrier to commercial deployment of GE wheat.     

小鼠ES细胞种系嵌合体的获得

种系嵌合体的获得是实现ＥＳ细胞介导的转基因途径的决定步骤,ＥＳ细胞种系分化能力的保持是决定种系嵌合的前提条件,而事体的主种系嵌合体的获得则是判定ＥＳ细胞系是否具有种系分化能力的唯一方法,为考察本室新近建立的３种小鼠ＥＳ细胞系ＭＥＳＰＵ２１．ＭＥＳＰＵ２２和ＭＥＳＰＵ２９的种系分化能力,选用近交系Ｃ５７ＢＬ／６Ｊ及远交系ＫＭＷ和ＩＣＲ为受体胚胎提供者,分别通过囊胚注射法和８细胞期桑椹胚注射法进行了嵌     

Genetic Screening for Meiotic Mutations of the Female Germline Mosaic Clones in Drosophila melanogaster

A method of screening for meiotic mutations based on genetic analysis of chromosome disjunction in germline mosaic clones of females homozygous for potential mutations is proposed. The clones are obtained at high frequency due to the use of the transgenic FLP/FRT system of mitotic recombination. This system permits obtaining homozygous clones in the first generation after mutagenesis, whereas the cultures are set up after selection for potential meiotic mutations. This significantly enhances, the efficiency of screening by the elimination of the limiting stage. Using this method, the following mutations were revealed in the 3L arm of Drosophila: ff6leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3leading to the formation of chromosome bridges in anaphase and telophase, chromosome nondisjunction, and premature chromatin condensation after metaphase; embryonic lethal ff29, with disturbed coordination between nuclear and centrosome cycles during syncytial cleavage; and a series of other mutations causing a wide spectrum of disturbances in male meiosis. Comparison of the proposed method with procedures of screening for yeast cell-cycle mutations showed that we succeeded in attaining the efficiency of screening in the Drosophilamodel close to that in the yeast model.     

The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.     

Molecular Spectrum of Spontaneous de Novo Mutations in Male and Female Germline Cells of Drosophila melanogaster

We carried out mutation screen experiments to understand the rate and molecular nature of spontaneous de novo mutations in Drosophila melanogaster, which are crucial for many evolutionary issues, but still poorly understood. We screened for eye-color and body-color mutations that occurred in the germline cells of the first generation offspring of wild-caught females. The offspring were from matings that had occurred in the field and therefore had a genetic composition close to that of flies in natural populations. We employed 1554 F₁ individuals from 374 wild-caught females for the experiments to avoid biased contributions of any particular genotype. From ~8.6 million alleles screened, we obtained 10 independent mutants: two point mutations (one for each sex), a single deletion of ~6 kb in a male, a single transposable element insertion in a female, five large deletions ranging in size from 40 to 500 kb in females, and a single mutation of unknown nature in a male. The five large deletions were presumably generated by nonallelic homologous recombination (NAHR) between transposable elements at different locations, illustrating the mutagenic nature of recombination. The high occurrence of NAHR that we observed has important consequences for genome evolution through the production of segmental duplications.     

Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

Stand Still, a Drosophila Gene Involved in the Female Germline for Proper Survival, Sex Determination and Differentiation

We identified a new gene, stand still (stil), required in the female germline for proper survival, sex determination and differentiation. Three strong loss-of-function alleles were isolated. The strongest phenotype exhibited by ovaries dissected from adult females is the complete absence of germ cells. In other ovaries, the few surviving germ cells frequently show a morphology typical of primary spermatocytes. still is not required either for fly viability or for male germline development. The gene was cloned and found to encode a novel protein. still is strongly expressed in the female germ cells. Using P[stil(+)] transgenes, we show that stil and a closely localized gene are involved in the modification of the ovarian phenotypes of the dominant alleles of ovo caused by heterozygosity of region 49 A-D. The similarity of the mutant phenotypes of stil to that of otu and ovo suggests that the three genes function in a common or in parallel pathways necessary in the female germline for its survival, sex determination and differentiation.     

The Use of Baker's Yeast in the Generation of Asymmetric Centers to Produce Chiral Drugs and Other Compounds

ABSTRACT:?

This review gives a general idea about the importance of chiral carbon in medicine and a way to obtain chiral building blocks with Baker's yeast (Saccharomyces cerevisiae) or synthesis of medicaments and other organic compounds. Reactions with these microorganisms are cheaper and easier to be executed than with chemicals (e.g., organometallics). Examples of important and practical reactions catalyzed by enzymes inside Saccharomyces cerevisiae are given and probable mechanisms of action of these enzymes are shown. Although these microbes have advantages such as low cost and availability, there are some concerns that are necessary to be resolved, such as NAD(P)H dosage to choose strains more adequate for reduction reactions.     

Genetic Characterization of Small Ovaries, a Gene Required in the Soma for the Development of the Drosophila Ovary and the Female Germline

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.     

Germline hypermutability in Drosophila and its relation to hybrid dysgenesis and cytotype

In its hypermutable state, an unstable singed allele, sn^w, mutates in the germline to two other alleleic forms at a total frequency usually between 40 and 60%. In its stable state, the mutation rate of sn^w is essentially zero. Its state depends on an extrachromosomal condition indistinguishable from a property called cytotype previously studied as a component of hybrid dysgenesis. Of the two known systems of hybrid dysgenesis, denoted P-M and I-R, sn^w hypermutability is determined by the P-M system and appears to be independent of the I-R system. Cytotype, as defined by the control of sn^w mutability, is self-reproducing in the cytoplasm or nucleoplasm of the germline through at least two generations. However, it is not entirely autonomous, being ultimately determined by the chromosomes after sufficiently many generations of backcrossing. This combination of chromosomal and extrachromosomal transmission agrees well with previous studies on cytotype. Temperature differences have little effect on the mean mutation rates, but they have a pronounced effect on the intrinsic variance among individuals. The latter effect suggests that high temperatures reduce germ-cell survival during the development of dysgenic flies. Chromosomal rearrangements produce no apparent effects on the behavior of sn^w. Hypermutability is thought to be caused by the excision or other alteration of an inserted genetic element in the sn^w gene. This element might be a copy of the "P factor," which is though to be a mobile sequence capable of causing female sterility and other dysgenic traits in the P-M system.     

Studies on Two Factors Affecting the Production of Germline Chimeras by Using HM 1 Cells

ＥＳ细胞系统与基因定位致变相结合,进行基因敲除（ｋｎｏｃｋｏｕｔ）已成为研究基因在生物体内功能的重要手段。在ＥＳ细胞系的建立、外源基因导入ＥＳ细胞、种系嵌合鼠的获得等三个重要环节中,种系嵌合鼠的获得是最关键的一环。由于ＥＳ细胞系统技术复杂、实验条件要求很高,尽管国际上已报导了上百例的基因敲除（ｋｎｏｃｋｏｕｔ）实验,但是到目前为止,我国还无一例在国内条件下获得种系嵌合鼠的正式报道。本研究对影响种系嵌合鼠获得的两种因素（饲养层细胞、受体胚胎种类）进行了比较研究,成功地获得了种系嵌合鼠。将ＨＭ１细胞在ＳＴＯ或ＭＥＦ培养层上培养至２１３３代,注射到不同小鼠的囊胚里,经过恢复培养,移植到假孕的昆明白雌鼠子宫内。由于ＨＭ１细胞来源于粟色的的１２９品系,而胚胎供体鼠的毛色为黑或白色,仔鼠出生一周后即可辨别是否为毛色嵌合鼠。用成年嵌合鼠与其受体胚胎相同品系的小鼠交配,进行种系嵌合鼠鉴定。曾有报导：ＳＴＯ培养层会导致ＥＳ细胞发生核变。我们改用ＭＥＦ培养层,获得嵌合鼠的比率高达４８．６％（Ｔａｂｌｅ１）。不同小鼠胚胎之间存在差异,Ｃ５７ＢＬ／６Ｊ、ＩＣＲ和昆明白三者提供的受体胚胎产生嵌合鼠的比率分别为７１．４％、５５％     

Characterization of IntA,a Bidirectional Site-Specific Recombinase Required for Conjugative Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42

Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.     

Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, P_attCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.     

Selection and Maintenance of Sexual Identity in the Drosophila Germline

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.     

A Regulatory Network of Drosophila Germline Stem Cell Self-Renewal

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A Germline Clone Screen for Meiotic Mutants in Drosophila melanogaster

Using a FLP/FRT-based method to create germline clones, we screened Drosophila chromosome arms 2L and 3R for new female meiotic mutants. The screen was designed to recover mutants with severe effects on meiotic exchange and/or segregation. This screen yielded 11 new mutants, including six alleles of previously known meiotic genes (c(2)M and ald/mps1). The remaining five mutants appear to define at least four new genes whose ablation results in severe meiotic defects. Three of the novel meiotic mutants were identified at the molecular level. Two of these, mcm5A7 and tremF9, define roles in meiotic recombination, while a third, conaA12, is important for synaptonemal complex assembly. Surprisingly, five of the nine mutants for which the lesion has been identified at the molecular level are not the result of mutations characteristic of EMS mutagenesis, but rather due to the insertion of the transposable element Doc. This study demonstrates the utility of germline clone-based screens for the discovery of strong meiotic mutants, including mutations in essential genes, and the use of molecular genetic techniques to map the loci.     

Use of Nystatin to Eliminate Spontaneous Revertants in Yeast

Studies of induction of suppressor mutants may be obscured by revertants already present in the treated population. Such revertants, which appear as a minority of prototrophs among auxotrophs, can be eliminated by pretreatment with nystatin before application of the mutagen. Treatment with nystatin, under the proper conditions, decreased the frequency of prototrophs about 30-fold. This is sufficient for studying induced mutation at frequencies close to the spontaneous frequency.     

雌性哺乳动物生殖干细胞：在争议中前进

100多年以来,雌性哺乳动物出生后是否存在生殖干细胞的争议尚无定论．2004年,研究人员从出生后的小鼠卵巢中发现并分离到雌性生殖干细胞(female germline stem cells,FGSCs),挑战了存在近半个世纪的理论：哺乳动物出生后不会对卵母细胞库进行更新．随后很多研究不仅指出哺乳动物出生后卵巢中新生成的卵母细胞源自FGSCs,而且发现如果将FGSCs移植回受体卵巢,它们能够产生功能性的卵母细胞并由此得到健康的后代．可是,有的研究小组重复实验或者精心设计实验,却未得到相同的结果,甚至得出相反的结果．最近,有研究者从育龄女性卵巢中分离到了在体内外都能够分化出功能性卵母细胞的FGSCs,不过这些卵母细胞的受精能力还有待证实．本文回顾了哺乳动物FGSCs的研究历程,并对这一存在已久的争论以及FGSCs研究方向和将来的运用前景展开了评述．