Mutant Rec-1 Eliminates the Meiotic Pattern of Crossing over in Caenorhabditis Elegans

Meiotic crossovers are not randomly distributed along the chromosome. In Caenorhabditis elegans the central portions of the autosomes have relatively few crossovers compared to the flanking regions. We have measured the frequency of crossing over for several intervals across chromosome I in strains mutant for rec-1. The chromosome is ~50 map units in both wild-type and rec-1 homozygotes, however, the distribution of exchanges is very different in rec-1. Map distances expand across the gene cluster and contract near the right end of the chromosome, resulting in a genetic map more consistent with the physical map. Mutations in two other genes, him-6 and him-14, also disrupted the distribution of exchanges. Unlike rec-1, individuals homozygous for him-6 and him-14 had an overall reduction in the amount of crossing over accompanied by a high frequency of nondisjunction and reduced egg hatching. In rec-1; him-6 and rec-1; him-14 homozygotes the frequency of crossing over was characteristic of the Him mutant phenotype, whereas the distribution of the reduced number of exchanges was characteristic of the Rec-1 pattern. It appears that these gene products play a role in establishing the meiotic pattern of exchange events.     

Purification and Characterization of Wild-Type and Mutant Tk1 Type Kinases From Caenorhabditis Elegans

Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ～60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher k_cat but unchanged K_m. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 μg/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability.     

Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination

A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.     

Effect of Different Sperm Numbers on Fertility after Artificial Insemination of Foxes

A total of 325 blue for vixens were inseminated with fresh semen from 50 silver fox males. Each ejaculutate was divided into 4 portions and diluted so as to contain 100, 60, 40, and 20 million sperm/ml. Vixens in groups 1,2,3 and 4 had been randomly assigned to their group at the time of insemination. The animals were inseminated once with either 100,60,40, or 20 million sperm. Vixens in groups 5 and 6 were selected by the technician after detecting signs of estrus during a physical examination. Animals judged to be at their optimal time for conception were assigned to group 5 and inseminated once with 20 million sperm. Animals considered to be early in their heat were assigned to group 6 and inseminated twice within 24 to 36 h with 20 million sperm per insemination dose. All inseminations were performed within 3 h of semen collection. A 1-ml total volume of extended semen was used for intrauterine deposition. In the random group inseminated once with 20 million sperm (group 4), both pregnancy rate and litter size were lower compared to the other random groups (groups 1,2, and 3), although the difference was not statistically significant. Among the vixens inseminated with 20 million sperm (group 4, 5, and 6) there was a significant difference in fertility between animals randomly selected and inseminaed once and those selected by the technician and inseminated twice (group 6). Our results suggest that for the crossbreeding of foxes 20 million sperm is the minimum insemination dose required for acceptable fertility with the present tecnique for sperm preservation and estrous determination.     

秀丽线虫的蛋白质组学研究

作为模式生物,秀丽线虫(Caenorhabditis elegans)已经成功地用于许多生命过程的研究,尤其被广泛应用于现代发育生物学、行为与神经生物学、基因组学、正向和反向的遗传学研究中,近年来,秀丽线虫更成为了一个进行蛋白质组学研究的优良体系,诠释了基于基因组学和RNA干涉研究中的基因功能。许多比较蛋白质组学表达谱的建立可以更好地理解线虫在不同发育阶段、不同温度下基因的表达,在与人类神经疾病相关的疾病研究中,线虫对帕金森疾病、阿尔茨海默症、衰老与寿命、胰岛素通路都有所揭示。另外,线虫的亚蛋白质组学和翻译后修饰如糖基化和磷酸化也已经鉴定,其数据库也在不断地完善。本文介绍了秀丽线虫的蛋白质表达谱建立的历史,尤其是神经科学研究中的应用及翻译后修饰表达谱的建立等方面的研究现状,因此,结合其它分子生物学和基因工程技术,线虫蛋白质组学研究已成为提供一个新的全面的系统分析基因功能的重要工具,提示线虫是"蠕虫蛋白质组学"的一个丰富宝藏。     

Macrorestriction Analysis of Caenorhabditis Elegans Genomic DNA

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a ``macrorestriction mapping' procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.     

Genetic Analysis of Defecation in Caenorhabditis Elegans

Defecation in the nematode Caenorhabditis elegans is achieved by a cyclical stereotyped motor program. The first step in each cycle is contraction of a set of posterior body muscles (pBoc), followed by contraction of a set of anterior body muscles (aBoc), and finally contraction of specialized anal muscles that open the anus and expel intestinal contents (Exp). By testing existing behavioral mutants and screening for new mutants that become constipated due to defects in defecation, I have identified 18 genes that are involved in defecation. Mutations in 16 of these genes affect specific parts of the motor program: mutations in two genes specifically affect the pBoc step; mutations in four genes affect the aBoc step; mutations in four genes affect the Exp step; and mutations in six genes affect both aBoc and Exp. Mutations in two other genes affect the defecation cycle period but have a normal motor program. Sensory inputs that regulate the cycle timing in the wild type are also described. On the basis of the phenotypes of the defecation mutants and of double mutants, I suggest a formal genetic pathway for the control of the defecation motor program.     

Sex Determination in Polyploids of Caenorhabditis Elegans

In Caenorhabditis elegans triploid animals with two X chromosomes (symbolized 3A;2X) are males. However, these triploid males can be feminized by making them mutant for recessive dosage compensation mutations, by adding X chromosome duplications or by microinjecting particular DNA sequences termed feminizing elements. None of these treatments affects diploid males. This study explores several aspects of these treatments in polyploids. The dosage compensation mutants exhibit a strong maternal effect, such that reduction of any of the dosage compensation gene functions in the mother leads to sex reversal of 3A;2X animals. Likewise, all X chromosome duplications tested cause both sex reversal and intersexual development of many 3A;2X animals. Microinjected feminizing element DNA does not cause extensive sex reversal, but does result in intersexual development in 3A;2X animals. Neither X chromosome duplications nor microinjected feminizing elements show the extreme maternal effect of the dosage compensation mutants, although there is indirect evidence for a maternal effect of the feminizing elements. In particular, very little feminizing element DNA needs to be microinjected in order to feminize triploid males, far less than what is needed for stable inheritance, implying that feminizing elements can work within the mother's gonad. However, even very high concentrations of microinjected feminizing elements do not affect sex determination in diploid males, suggesting that they are not part of the numerator of the X/A ratio. In addition, no pair of X chromosome duplications feminizes diploid males, suggesting that none of these duplications contains a numerator of the X/A ratio. Instead, I infer that an X-linked locus, as yet undefined, must be present in two copies for hermaphrodite development to ensue or that the two X chromosomes might interact.     

Mutations Affecting the Chemosensory Neurons of Caenorhabditis Elegans

We have identified and characterized 95 mutations that reduce or abolish dye filling of amphid and phasmid neurons and that have little effect on viability, fertility or movement. Twenty-seven mutations occurred spontaneously in strains with a high frequency of transposon insertion. Sixty-eight were isolated after treatment with EMS. All of the mutations result in defects in one or more chemosensory responses, such as chemotaxis to ammonium chloride or formation of dauer larvae under conditions of starvation and overcrowding. Seventy-five of the mutations are alleles of 12 previously defined genes, mutations which were previously shown to lead to defects in amphid ultrastructure. We have assigned 20 mutations to 13 new genes, called dyf-1 through dyf-13. We expect that the genes represented by dye-filling defective mutants are important for the differentiation of amphid and phasmid chemosensilla.     

Caenorhabditis Elegans Mutants Resistant to Inhibitors of Acetylcholinesterase

We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions.     

Molecular Genetics of the Caenorhabditis Elegans Heterochronic Gene Lin-14

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.     

Genetic Organization of the Unc-60 Region in Caenorhabditis Elegans

We have investigated the chromosomal region around unc-60 V, a gene affecting muscle structure, in the nematode Caenorhabditis elegans. The region studied covers 3 map units and lies at the left end of linkage group (LG) V. Compared to the region around dpy-11 (at the center of LGV), the unc-60 region has relatively few visible genes per map unit. We found the same to be true for essential genes. By screening simultaneously for recessive lethals closely linked to either dpy-11 or unc-60, we recovered ethyl methanesulfonate-induced mutations in 10 essential genes near dpy-11 but in only two genes near unc-60. Four deficiency breakpoints were mapped to the unc-60 region. Using recombination and deficiency mapping we established the following gene order: let-336, unc-34, let-326, unc-60, emb-29, let-426. Regarding unc-60 itself, we compared the effect of ten alleles (including five isolated during this study) on hermaphrodite mobility and fecundity. We used intragenic mapping to position eight of these alleles. The results show that these alleles are not distributed uniformly within the gene, but map to two groups approximately 0.012 map unit apart.     

The Ncl-1 Gene and Genetic Mosaics of Caenorhabditis Elegans

A ncl-1 mutation results in enlarged nucleoli, which can be detected in nearly all cells of living animals by Nomarski microscopy. Spontaneous mitotic loss of a ncl-1(+)-containing free duplication in an otherwise homozygous ncl-1 mutant animal results in mosaicism for ncl-1 expression, and the patterns of mosaicism lead us to conclude that ncl-1 acts cell autonomously. The probability of mitotic loss of the duplication sDp3 is approximately constant over many cell divisions. About 60% of the losses of sDp3 at the first embryonic cell division involve nondisjunction. Frequencies of mitotic loss of different ncl-1(+)-bearing free duplications varied over a 200-fold range. The frequencies of mitotic loss were enhanced by a chromosomal him-10 mutation. We have used ncl-1 as a cell autonomous marker in the mosaic analysis of dpy-1 and lin-37. The focus of action of dpy-1 is in hypodermis. A mutation in lin-37 combined with a mutation in another gene results in a synthetic multivulva phenotype. We show that lin-37 acts cell nonautonomously and propose that it plays a role, along with the previously studied gene lin-15, in the generation of an intercellular signal by hyp7 that represses vulval development.     

秀丽线虫脂肪积累调控的生理与分子机制

脂肪积累是一个复杂的生理过程,模式动物秀丽线虫(以下简称线虫)已经成为目前研究脂肪积累的重要模型.线虫中的脂肪酸代谢通路与其他物种中的代谢是基本一致的,很多关键的代谢调节基因的功能已经得到鉴定.线虫中脂肪积累涉及至少4个核心调控通路,分别为胰岛素和转化生长因子β(TGF-β)信号通路、sbp-1/ mdt-15介导的信号通路、核激素受体nhr-49介导的信号通路与雷帕霉素靶标(TOR)和氨基己糖介导的信号通路.此外,神经递质5-羟色胺、多巴胺和谷氨酸参与了脂肪积累的调控,而tub-1和bbs-1可以介导对脂肪积累的神经调控,暗示了纤毛结构与感觉神经元在脂肪积累中可能的重要功能.线虫中的研究工作对人类肥胖症等代谢疾病的研究具有重要的提示作用.     

The Meiotic Behavior of an Inversion in Caenorhabditis Elegans

The rearrangement hIn1(I) was isolated as a crossover suppressor for the right end of linkage group (LG) I. By inducing genetic markers on this crossover suppressor and establishing the gene order in the homozygote, hIn1(I) was demonstrated to be the first genetically proven inversion in Caenorhabditis elegans. hIn1(I) extensively suppresses recombination in heterozygotes in the right arm of chromosome I from unc-75 to unc-54. This suppression is associated with enhancement of recombination in other regions of the chromosome. The enhancement observed maintains the normal distribution of events but does not extend to other chromosomes. The genetic distance of chromosome I in inversion heterozygotes approaches 50 map units (m.u.), approximately equal to one chiasma per meiosis. This value is maintained in hIn1(I)/szT1(I;X) heterozygotes indicating that small homologous regions can pair and recombine efficiently. hIn1(I)/hT2(I;III) heterozygotes share no uninverted homologous regions and segregate randomly, suggesting the importance of chiasma formation in proper segregation of chromosomes. The genetic distance of chromosome I in these heterozygotes is less that 1 m.u., indicating that crossing over can be suppressed along an entire chromosome. Since one of our goals was to develop an efficient balancer for the right end of LGI, the effectiveness of hIn1(I) as a balancer was tested by isolating and maintaining lethal mutations. The meiotic behaviour of hIn1(I) is consistent with other genetic and cytogenetic data suggesting the meiotic chromosomes are monocentric. Rare recombinants bearing duplications and deficiencies of chromosome I were recovered from hIn1(I) heterozygotes, leading to the proposal the inversion was paracentric.     

Natural Variation and Copulatory Plug Formation in Caenorhabditis Elegans

Most of the available natural isolates of the nematode Caenorhabditis elegans have been examined and compared with the standard laboratory wild type (Bristol N2). Molecular markers, in particular transposon restriction fragment length polymorphisms, were used to assign these isolates to 22 different races, for which brood size and spontaneous male frequency were determined. Several distinctive traits were observed in some of these races. One example is mab-23, in a race from Vancouver, which leads to severe distortion of male genitalia and prevents male mating. Another is gro-1, segregating in a Californian race, which is associated with slow growth, heat resistance and longevity. Many races differ from N2 in carrying a dominant allele at the plg-1 locus, causing copulatory plug formation by males. Properties and possible advantages of the plugging trait have been investigated. The dominant plg-1 allele does not lead to increased male mating efficiency, but males from a Stanford race (CB4855), in which the plugging trait was first observed, are much more virile than N2 males. Crosses between N2 and CB4855 indicate that the higher virility is due to multiple factors. Size differences between N2 and CB4855 are associated with factors mapping to LGV and LGX.