Host-selection behaviour by genetically identical aphids with different plant preferences

The behaviour of summer and autumn winged forms of the black bean aphid, Aphis fabae Scopoli (Homoptera: Aphididae), was compared on two plants utilized at different stages of the insect’s life cycle. Adult autumn migrants (gynoparae) are monophagous, colonizing spindle (Euonymus europaeus), whereas polyphagous summer winged aphids (alate virginoparae) are associated with a variety of herbaceous plants, including broad bean (Vicia faba). When aphids from a single clone were given access to a spindle leaf and a bean seedling in choice tests, many virginoparae settled and larviposited on both plant species over 24 h. By contrast, gynoparae showed a clear preference for spindle, with 93.5% of settled adults and 98.3% of larvae on this plant species. Close‐up video monitoring showed that gynoparae discriminated beans from spindle within a 5‐min period, whereas virginoparae behaved similarly on both plant species. For gynoparae, the major behavioural difference on the two plants appeared after a brief (epidermal) stylet penetration, with many insects taking flight within a few seconds of stylet withdrawal from bean. Factors detected during stylet insertion by gynoparae must therefore inhibit take‐off on spindle. Electrical recording experiments showed that aphids often punctured a cell membrane during brief probes on both plant species, and intracellular stylet activities always included a waveform associated with ingestion. When gynoparae puncture spindle cells their behaviour is probably modified by intracellular metabolites detected via gustation of ingested epidermal cell sap. These cues may inhibit the take‐off reflex which otherwise follows probing.     

Semen characteristics of genetically identical quadruplet bulls

Modern cloning methods have become an important technology in artificial insemination which is used to create and maintain pools of genetically superior bull semen. In this study, semen from four identical quadruplet bulls (Q(1), Q(2), Q(3), and Q(4)) produced by blastomere separation was analyzed to evaluate the differences in reproductive potential, if any, that existed between the identical quadruplet siblings. Analysis of fresh semen collected from 1994 to 1996, showed lower progressive motility and lower sperm concentration for one bull (Q(3)) compared to his identical brothers (P<0.05). Semen characteristics following freezing-thawing procedures have also been tested for these quadruplet bulls. The percentage of motility, progressive motility, and mean path velocity were lower in Q(4) compared with Q(1). Moreover, intracellular calcium level and P25b level (P25b is a sperm surface protein proposed to be a potential bull fertility marker) were lower in Q(4) compared with his siblings (P<0.05). Cryodamage to Q(4)'s frozen-thawed spermatozoa were confirmed by a lower percentage of embryo development after in vitro fertilization. Thus, the higher instability of cryopreserved spermatozoa from Q(4) and the lower semen production of Q(3), compared to their siblings, indicate that differences in semen characteristics can indeed exist among genetically identical animals produced by blastomere separation.     

Production of monozygotic (identical) horse twins by embryo micromanipulation

The blastomeres of 192- to 8-cell embryos recovered surgically 1-3 days after ovulation from 23 Pony mares were mechanically separated and inserted, in various combinations, into evacuated pig zonae pellucidae to make 27 'half' and 17 'quarter' micromanipulated embryos. These were embedded in agar and cultured in vivo in the ligated oviducts of ewes for 3.5-5 days to allow development to the late morula/early blastocyst stage. Subsequent surgical or non-surgical transfer of 13 'half' and 17 'quarter' embryos to mares resulted in 10 established pregnancies, including 2 monozygotic pairs. Surgical transfer to mares that had not been recently used as donors of embryos was more successful (10/20) than surgical or non-surgical transfer to recently operated mares (0/10).     

Semen and reproductive profiles of genetically identical cloned bulls

In this comparative study, reproductive parameters and semen characteristics of cloned bulls (n = 3) derived from somatic cell nuclear transfer (SCNT) were compared to their original cell donor Holstein-Friesian (n = 2) bulls from the same enterprise to assess the differences in reproductive potential between a donor bull and its clones. The parameters evaluated included motility of fresh, frozen-thawed and Percoll-treated frozen-thawed spermatozoa, as well as in vitro fertilization (IVF) ability, embryo quality, birth and survival of calves following IVF and embryo transfer with frozen-thawed semen. With fresh semen, spermatozoa from one cloned bull had lower motility than its donor. Cloned bulls had higher velocity parameters in fresh semen, but those effects were not obvious in frozen-thawed or frozen-thawed semen selected with a Percoll gradient. Semen collected from cloned bulls had significantly higher IVF rates compared to donors; however, embryo development per cleaved embryo or quality of blastocysts did not differ between donors and cloned bulls. Pregnancy and live offspring rates from one donor and its cloned bull did not differ between fresh (40%, 16/40 versus 46%, 17/37) and vitrified/thawed (13%, 2/16 versus 25%, 4/16) embryo transfer following IVF. A total of 26 calves were obtained from genotypically identical donor and cloned bulls with no signs of phenotypical abnormalities. These preliminary results suggested that the physiology of surviving postpubertal cloned bulls and quality of collected semen had equivalent reproductive potential to their original cell donor, with no evidence of any deleterious effects in their progeny.     

Ethanol production by genetically modified strains of Saccharomyces

Summary Two polyploid yeast strains and two genetically manipulated yeast strains were subjected to anaerobic fermentations in whole corn mash and defined media. Carbohydrate utilization and ethanol production rates were investigated. Whilst the polyploid strains exhibited superior performance in the whole corn mash, the genetically manipulated strains were so in defined media with glucose as the substrate. The overall fermentation performance of the novel strains however was comparable to the polyploid strains with corn mash as the substrate when most of the solid material had been removed. The flocculating and dextrin utilizing properties of the yeast strains examined play an important role in such fermentations.     

Production of genetically identical swine: Uses for families with reduced phenotypic variation

Recent advances in reproductive technology hold promise for future production of nearly unlimited numbers of swine with identical genotypes through the use of nuclear transfer. Potential benefits of use of these animals in the swine industry and in research are examined in the present study. Variability of litter size, days to slaughter and backfat thickness are predicted for clones, full-siblings and unrelated animals. One hundred simulation runs of 100 unrelated animals and 25 families of 4 full-siblings and clones were completed for each trait. Expected phenotypic standard deviations were 2.5 pigs for litter size, 12 d for days to slaughter and 2 mm for backfat thickness. Between family variances were expected to include 50% additive genetic variance, 25% dominance variance and 100% common environmental variance for full-siblings and 100% of all 3 components for clones. Substantial advantages of clones over unrelated animals and smaller advantages of clones over full-siblings were found for variability of days to slaughter and backfat thickness, but little difference was found for litter size. Ratios of error mean squares from analyses of variance suggest that use of clones rather than unrelated animals could reduce the number of animals needed for experiments by 67 and 65% for days to slaughter and backfat thickness, respectively, but only by 12% for litter size. These results suggest that the first use of cloned swine would be to reduce the numbers of research animals needed for intensive studies.     

Nuclear transplantation as a method of producing genetically identical livestock

Abstract

Genetic variation is a problem faced by both researchers and producers. One method to reduce genetic variation is to clone embryos by nuclear transfer. Implementation, involves transferring nuclei from a morula stage embryo to unfertilized oocytes from which the metaphase II chromosomes have been removed. Since each of the nuclei from the original morula stage embryo are genetically identical, each of the embryos that are a result of nuclear transfer have identical nuclear genetics. The original morula stage embryo, relatively speaking, is more differentiated than a one‐cell stage embryo. Thus for the resulting nuclear transfer embryo to continue in development the transferred nuclei must be remodeled to resemble nuclei of a one‐cell stage embryo and be reprogrammed in their developmental cascade of events to behave as nuclei of a one‐cell stage embryo. The potential applications of producing genetically identical individuals range from reducing the number of animals needed for experimentation to providing a more uniform product in the freezer at the grocery store. Unfortunately, the procedures for producing cloned animals by nuclear transfer are still relatively inefficient. There is a need for more basic research to be conducted in understanding mammalian embryogenesis for the application of this and other biotechnologies.     

The effects of prolonged coffee intake on genetically identical mice

C57BL6J mice were fed coffee over a prolonged period of time (adult life). They had a statistically significant decrease in longevity as compared to the genetically identical controls. The coffee fed animals developed a markedly deteriorated physical condition and lower weights despite the fact that they consumed as much or more food and fluid than the water fed controls.     

Development and phenotypic variability of genetically identical half mouse embryos

The growth and development of three groups of genetically identical F1 C57BL/6J female x SJL/J male mice were compared to examine whether embryo manipulation affects subsequent postnatal growth and development of mammalian embryos: (1) controls--the natural offspring of timed matings, (2) transferred controls--offspring from 2-cell embryos transferred to recipients 1 day asynchronous, and (3) transferred half embryos--offspring developing from one blastomere from the 2-cell stage transferred to recipients 1 day asynchronous. The recipients were C57BL/6J females. No differences were found in the age at eye opening and vaginal opening. At 5 days after birth the median body weights of the controls were lower than the weights of the transferred groups. This result could be explained by the larger litter size in the control group. The overall variances of the body weights did not differ between the groups. By the second week after birth a marked increase in overall variances of body weights of the transferred groups, compared with the control group, was observed. At 5 days after birth, the median tail lengths did not differ between groups, and overall variances were the same. By the second week, the overall variances of the tail lengths of the transferred groups were significantly greater than that of the control group. Possibly the increased overall variances of the body weight and the tail length of the transferred groups are related to the smaller litter size in these groups which affects competition for food and the ambient temperature in the nest. The overall results suggest newborn mice that have developed from half embryos have compensated for their initial deficiency. The intraclass correlation coefficients for body weight and tail length are approximately the same in all groups. Thus, producing artificial identical twins by embryo bisection may not affect their potential usefulness in the design of experiments.     

Phenotypic variation in a genetically identical population of mice.

The parental alleles of an imprinted gene acquire their distinctive methylation patterns at different times in development. For the imprinted RSVIgmyc transgene, methylation of the maternal allele is established in the oocyte and invariably transmitted to the embryo. In contrast, the methylation of the paternal allele originates during embryogenesis. Here, we show that the paternal methylation pattern among mice with identical genetic backgrounds is subject to extensive variation. In addition to this nongenetic variation, the process underlying RSVIgmyc methylation in the embryo is also subject to considerable genetic regulation. The paternal transgene allele is highly methylated in an inbred C57BL/6J strain, whereas it is relatively undermethylated in an inbred FVB/N strain. Individual methylation patterns of paternal alleles, and therefore all of the variation (nongenetic and genetic) in methylation patterns within an RSVIgmyc transgenic line, are established in early embryogenesis. For each mouse, the paternal RSVIgmyc allele is unmethylated at the day-3.5 blastocyst stage, and the final, adult methylation pattern is found no later than day 8.5 of embryogenesis. Because of the strong relationship between RSVIgmyc methylation and expression, the variation in methylation is also manifest as variation in transgene expression. These results identify embryonic de novo methylation as an important source of both genetic and nongenetic contributions to phenotypic variation and, as such, further our understanding of the developmental origin of imprinted genes.     

Centrosome splitting during nuclear elongation in the Drosophila embryo

In the early Drosophila embryo, nuclear elongation occurs during cellularization of the syncytial blastoderm. This process is closely related to the presence of microtubular bundles forming a basket-like structure surrounding the nuclei. In immunofluorescence observations with antibodies against alpha-tubulin, the microtubules appear to radiate from two bright foci widely separated from each other. We used electron microscopy to show that these foci are true centrosomes constituted by daughter and parent centrioles orthogonally disposed and surrounded by pericentriolar electrondense material. The centrosomes may be observed in the apical region of the blastoderm cells from the beginning of cellularization until the reestablishment of the first postblastodermic mitosis, when they organize the spindle poles. Until this time the dimensions of the procentrioles remain unchanged. The significance of these results is discussed in relation to the known behavior of centrioles in the cell cycle.     

Body handedness is directed by genetically determined cytoskeletal dynamics in the early embryo

Although substantial progress has been made recently in understanding the establishment of left-right asymmetry in several organisms, little is known about the initial step for any embryo. In gastropods, left-right body handedness is determined by an unknown maternally inherited single gene or genes at closely linked loci and is associated with the sense of spiral cleavage in early embryos. Contrary to what has been believed, we show that temporal and spatial cytoskeletal dynamics for the left- and right-handed snails within a species are not mirror images of each other. Thus, during the third cleavage of Lymnaea stagnalis, helical spindle inclination (SI) and spiral blastomere deformation (SD) are observed only in the dominant dextral embryos at metaphase-anaphase, whereas in the recessive sinistral embryos, helicity emerges during the furrow ingression. Actin depolymerization agents altered both cleavages to neutral. Further, we found a strong genetic linkage between the handedness-specific cytoskeletal organization and the organismal handedness, using backcrossed F4 congenic animals that inherit only 1/16 of dextral strain-derived genome either with or without the dextrality-determining gene(s). Physa acuta, a sinistral-only gastropod, exhibits substantial SD and SI levotropically. Thus, cytoskeletal dynamics have a crucial role in determination of body handedness with further molecular, cellular, and evolutionary implications.     

枯草芽孢杆菌基因修饰生产核黄素

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。     

Potential application of genetically identical somatic cell nuclear transfer-cloned dogs for gastrointestinal microbiota analysis

This study investigated the gastrointestinal microbiota in three genetically identical cloned dogs (A, B and C) by somatic cell nuclear transfer. We collected feces from three cloned dogs and their feed to investigate gastrointestinal microbiota using both culture-dependent and culture-independent methods. A total of 962 strains from the feces of cloned dogs were isolated using aerobic and anaerobic culture methods. The dominant microorganisms were Enterococcus faecalis and Enterococcus faecium in all fecal samples. In particular, the fecal sample from cloned dog C had similar proportions of three species (E. faecalis, E. faecium and Lactobacillus murinus). In all, 29 DNA fragments were identified by PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The highest DGGE band intensities were for E. faecalis from cloned dogs A and C and for Clostridium sordellii from cloned dog B, with relative intensities of 15.2, 17.7 and 14.4%, respectively. The other strains identified from the cloned dogs were Chryseobacterium soldanellicola, Escherichia coli, L. murinus, Streptococcus alactolyticus, Weissella confusa and uncultured bacterium. Some microbes isolated from the fecal samples, including C. soldanellicola and W. confuse, were derived from the feed. Overall, gastrointestinal microbiota of all genetically identical cloned dogs, maintained under the same environmental and feeding conditions, showed similar profiles in terms of species diversity analyzed by PCR-DGGE, although there were proportional differences in the amounts of bacterial species. To our knowledge, this is the first report to investigate and compare gastrointestinal microbiota of three genetically identical dogs.     

Variability of autogamy-maturation pattern in genetically identical populations of Paramecium tetraurelia

Autogamy in Paramecium tetraurelia is a form of sexual reproduction in a single cell that results in homozygosity in every genetic locus. Autogamy becomes inducible by natural starvation several fissions after the previous autogamy, and percent autogamy increases gradually with clonal age to reach 100%. We here report the degree of variability of the autogamy-maturation pattern, and how it is inherited through autogamous generations. We assessed the autogamy-maturation pattern by monitoring percent autogamy at the ages of 9, 18 and 27 fissions in the wild-type stock 51. To determine how the autogamy-maturation pattern is inherited, clones that showed the lowest and the highest percent autogamy at age 18 in a given autogamous generation (Gn) were examined for their percent autogamy in the next autogamous generation (Gn+1). This procedure was repeated through successive autogamous generations. We found that percent autogamy at ages 9 and 27 was rather stable (low and high, respectively), while it was extremely variable at age 18 ranging from 3% to 100%. We also found that percent autogamy at age 18 in the progeny clones was variable irrespective of percent autogamy at age 18 in the parental clones; there was no regular rule such as producing progeny with higher (or lower) percent autogamy from parents with lower (or higher) percent autogamy.     

The production of transgenic mice by embryo microinjection

The production of transgenic mice is a technology of great utility in the dissection of complex biological processes. This article is intended as a detailed primer for people interested in learning to produce transgenic mice, and discusses equipment, methods, and future directions for this technique.     

Development of efficient strategies for the production of genetically modified pigs

Although pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs, the efficiency of production of transgenic offspring is generally plagued by 1% of the DNA-injected embryos. Therefore, a problem with this method is the need for large numbers of pronuclear stage embryos. One great advancement would be the use of in vitro-matured (IVM) oocytes for the purpose of transgenic pig production. High developmental competence of IVM oocytes was proven by transfer of parthenogenetic IVM oocytes. A combined method of sperm vectors with the IVM of oocytes would make the production of transgenic pigs remarkably feasible. Rate of blastocyst formation following intracytoplasmic sperm injection (ICSI) by frozen sperm was over 20%, and transgene was expressed in approximately 50% of blastocysts generated. Somatic cell nuclear transfer would enable more efficient and sophisticated genetic modification of the pig. Simultaneous comparison between two nuclear transfer methods by electro-fusion and intracytoplasmic injection revealed clear differences in the pattern of nuclear remodeling and development of the reconstructed embryos. To specify the donor cell type that allows efficient genetic modification and easy reprogramming or to establish such cell lines is a critical issue in pig cloning. We tested pre-adipocytes from the subcutaneous adipose tissue of adult pigs for nuclear transfer. Cell cycle synchronization by differentiation induction is unique to the pre-adipocytes. Frequency of apoptosis was low in the cells synchronized by differentiation induction compared with other synchronization methods, including serum starvation, confluency, and chemical treatment. It would be of great worth if cryopreserved clone embryos were available. We have demonstrated that cryopreservation of in vitro-produced porcine embryos as well as clone blastocysts is possible by our unique method.     

Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l⁻¹ during shake flask conditions. A volume-corrected concentration of 11.3 g l⁻¹ of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol.