The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Studies on radiation-induced chromosome aberrations in mouse spermatocytes. II. Dose-response relationships of chromosome aberrations induced at zygotene stage in mouse primary spermatocytes following fast neutron- and 60Co gamma-irradiations

The chromosome aberrations induced at zygotene stage in mouse spermatocytes following exposures to fast neutrons and 60Co gamma-rays were examined at diakinesis-metaphase I. The dose-response relationships were well fitted to linear equation for deletion-type aberrations and to linear-quadratic equation for exchange-type aberrations in 60Co gamma-irradiation group. In fast neutron-irradiation group, the dose-response relationships were well fitted to linear equations for deletion- and exchange-type aberrations. The rate of deletion-type aberrations was remarkably high for fast neutrons, about 6 times higher than that after 60Co gamma-irradiation. The main types of chromosome aberrations observed were iso-chromatid breaks or fragments and chromatid exchanges in both irradiation groups as well as X-irradiation. These results indicate that there is a possibility that two double-strand breaks are induced simultaneously at iso-locus position in sister chromatids by a single track of radiations. Production of such single-track-induced two double-strand breaks in iso-chromatids may be very frequently expressed as iso-chromatid-type deletions in the high LET fast neutron-irradiation group. On the contrary, in the low LET 60Co gamma- or X-irradiation group, the above-mentioned mechanism may not be so effective for contribution to chromosome aberration induction in mouse spermatocytes. This mechanism was discussed in detail.     

Sex chromosome configurations in pachytene spermatocytes of an XYY mouse

Karyotypic investigation of a phenotypically normal but sterile male mouse showed the presence of an XYY sex chromosome constitution. The synaptic behaviour of the three sex chromosomes was examined in 65 pachytene cells. The sex chromosomes formed a variety of synaptic configurations: an XYY trivalent (40%); an XY bivalent and Y univalent (38.5%); an X univalent and YY bivalent (13.8%); or X, Y, Y univalence (7.7%). There was considerable variation in the extent of synapsis and some of the associations clearly involved nonhomologous pairing. These observations have been compared with previously published information on chromosome configurations at metaphase I from other XYY males.     

Expression of tumor necrosis factor at a specific developmental stage of mouse embryos

We investigated the expression of the tumor necrosis factor (TNF) gene during development of mouse embryos, and observed its transient expression on Days 9 and 10 of gestation. We also detected a 25-kDa protein showing immunological cross-reactivity with mouse TNF antibody in an extract of 10-day embryos. These results suggest that TNF plays a role in mammalian ontogenesis.     

茎瘤芥不同生长期植株营养特性及其与产量的关系

在涪陵区选取30个茎瘤芥种植农户,采用大田调查和室内化学分析方法,研究了茎瘤芥不同生长期（苗期、快速膨大期、采收期）叶片和茎瘤10种必需营养元素含量的变化特征及其与产量的关系。结果表明：茎瘤芥在整个生育期内,除K、S含量较高外,其余大、微量元素均在大多植物含量范围内;不同生育期茎瘤芥叶片、茎瘤中各养分含量变化具有明显的规律性,苗期叶片大量元素含量次序为N>K>Ca>P>S>Mg,快速膨大期和采收期叶片大量元素含量次序均为N>K>Ca>S>P>Mg,茎瘤中大量元素含量次序均为K>N>P>S>Ca>Mg,3个生长期叶片和茎瘤的微量元素含量,除快速膨大期茎瘤中略有不同（Fe>Zn>Mn>Cu）外,其余均为Fe>Mn>Zn>Cu;从苗期到快速膨大期再到采收期养分变化规律看,叶片中N、P、K、Fe、Cu和Zn含量呈降低趋势,而Ca、Mg、S和Mn则呈现先降低后升高的趋势,从快速膨大期到采收期茎瘤中除N、S、Fe和Cu元素呈降低趋势外,其余养分元素均呈上升趋势。从茎瘤芥不同器官养分含量高低看,快速膨大期和采收期叶片中N、P、K、Cu和Zn含量较茎瘤中低,而Ca、Fe和Mn含量的变化特点则相反,S和Mg差异较小,表明茎瘤芥不同部位对不同养分的敏感程度各异。相关分析表明,各生育期不同器官的Mg、Fe、Mn和Zn与产量呈显著或极显著的负相关关系,K、Cu与产量呈正的相关关系。通过逐步回归分析建立茎瘤芥各生育期植株营养元素与产量的回归预测模型,其中苗期叶片营养元素与产量的最优回归方程为Y= 36768 3583XK-6.328XFe-76.09XMn;快速膨大期叶片和茎瘤营养元素与产量的最优回归方程分别为Y=50458 21557XP 7925XCa-88092XMg-1145XCu和Y=32487 7294XK-116122XMg;采收期叶片和茎瘤营养元素与产量的最优回归方程分别为Y=36064 3413XK-30.15XFe和Y= 11791 7334XK-385XZn。因此,在茎瘤芥各生长期均应注意钾肥的合理施用,快速膨大期应重视磷肥的施用。而几种微量元素和镁素对茎瘤芥产量的负效应,则可通过增施充分腐熟的有机肥料加以调控。     

A distinct FMRP polysomal population at an advanced stage of mammalian erythropoiesis

The fragile-X syndrome is a mental disorder caused by the absence of FMRP (the Fragile-X Mental Retardation Protein). While FMRP is found to be associated with the ribosomal components, its precise translational function remains to be defined. Here we report that FMRP is not found with the abundant free polysomes of the reticulocyte lysate, but rather with a heavy ribonucleoprotein complex sedimenting over 400S. This unusual distribution of FMRP at an advanced stage of mammalian erythropoiesis may unveil the discrete role of FMRP in translation.     

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.     

ADAM12‐deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects

ADAM (a d isintegrin a nd m etalloprotease) constitutes a family of multi‐domain proteins that are involved in development, homeostasis, and disease. ADAM12 plays important roles in myogenesis and adipogenesis in mice; however, the precise physiological mechanisms are not known, and the function of this gene in other vertebrates has not been examined. In this study, we used a simple model vertebrate, the zebrafish, to investigate the functions of ADAM12 during development. Zebrafish adam12 is conserved with those of mammals in the synteny and the amino‐acid sequence. We examined adam12 expression in zebrafish embryos by whole mount in situ hybridization and the promoter activity of the adam12 upstream sequence. We found that adam12 is strongly expressed in the cardiovascular system, erythroid progenitors, brain, and jaw cartilage during zebrafish development, and adam12‐knockout zebrafish exhibited reduced body size in the juvenile stage without apparent morphological defects. Taken together, these results suggest that adam12 plays a significant role in the regulation of body growth during juvenile stage in zebrafish, although the precise molecular mechanisms await further study.     

Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure

The fertilized mouse egg actively demethylates the paternal genome within a few hours after fertilization, whereas the maternal genome is only passively demethylated by a replication-dependent mechanism after the two-cell stage. This evolutionarily conserved assymetry in the early diploid mammalian embryo may have a role in methylation reprogramming of the two very different sets of sperm and egg chromatin for somatic development and formation of totipotent cells. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed that the incidence of abnormal methylation patterns differs between mouse two-cell embryos from superovulated females, nonsuperovulated matings, and in vitro fertilization (IVF). It also depends on embryo culture conditions and genetic background. In general, there was a good correlation with the number of embryos (from the same experiment) which did not develop in vitro up to the blastocyst stage. Thus, aberrant genome-wide DNA methylation in early embryos may be an important mechanism contributing to the high incidence of developmental failure in mammals. Similar to the situation in abnormally methylated embryos from nuclear transfer, it may cause a high incidence of pregnancy loss and abnormal phenotypes.     

Biosynthesis of a rare di-N-acetylated sugar in the lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis occurs via an identical scheme despite different gene clusters

Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD, and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB to wbpI were present, but a putative dehydrogenase gene was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Nonpolar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO(1629) and wbpO(3150), were shown to complement a wbpA knockout of P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO(1629) and WbpO(3150), and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, UDP-D-GalNAc, to a lesser extent, and UDP-D-Glc, to a much lesser extent. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed for P. aeruginosa, despite differences in the genomic context of the genes involved.     

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1–7 days of differentiation), mid-stage EBs groups (9–15 days of differentiation), maturing EBs groups (17–45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.     

春小麦不同发育阶段抗氧化系统对田间缓慢干旱的响应

研究了两种抗旱性不同的春小麦(Triticum aestivum L.)品种定西24(抗旱性较强)和品种8139(抗旱性较弱)不同发育阶段体内抗氧化系统对田间缓慢干旱的响应情况。结果表明,在田间自然干旱条件下,随着土壤水分含量的逐渐降低,两品种小麦叶片水势和含水量均缓慢降低,叶片色素及可溶性蛋白含量也于幼苗期显著降低,植株生长受抑。叶片抗氧化酶系统如SOD、：POD、CAT以及GSH—ASC循环中的两种关键酶APX和GR活性,均在小麦发育前期如幼苗期和拔节期显著升高而于后期下降。主要抗氧化物质ASC含量也表现出相似的变化趋势。虽然抗氧化系统在两春小麦品种不同发育阶段对田间干旱的响应行为大体相同,而且干旱较敏感品种8139各物质对干旱的响应强于干旱较耐受品种定西24,但后者减轻干旱导致的氧化损伤的效率高于前者。试验还表明,在作物的不同发育阶段,抗氧化系统对田间缓慢干旱响应的策略不同,前期如幼苗期和拔节期主要表现为积极应对,后期如抽穗期和灌浆期主要表现为被动忍耐。     

Comparative studies on the mRNA expression of development-related genes in an individual mouse blastocyst with different developmental potential

The evaluation of embryo morphology, widely used for selecting mammalian embryos before transfer, is not an adequate standard for selecting nuclear-transferred (NT) embryos. To search for markers useful for predicting the potential of NT embryos to develop into young, we examined the relation between the morphology of embryos with different developmental potential and gene expression of Oct 4, Nanog, Stat3, FGF4, Stella, and Sox2. In the present study, we examined pronuclear-exchanged blastocysts and morula blastomere, embryonic stem (ES) cell, and cumulus cell NT blastocysts, and in vivo-developed and in vitro-developed blastocysts. Based on the small variations in the gene expression levels among the in vivo-developed blastocysts, and the significant differences in gene expression between in vivo-developed (high developmental potential), and ES cell and cumulus cell NT blastocysts (low developmental potential), down-regulation of Sox2 and Oct4 genes is considered to be a candidate marker for the low potential of NT embryos to develop into young.     

Distinct functions of MLH3 at recombination hot spots in the mouse

The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1-MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that Mlh3(-/-) exhibit a 85-94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in Mlh3(-/-) meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in Mlh3(-/-) spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that approximately 10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals.     

Pattern of ribonucleic acid synthesis in vitro in primary spermatocytes from mouse testis carrying an X-autosome translocation

In an attempt to elucidate the mechanism of sterility of X-autosome translocations in the mouse, we studied the distribution of [³H]-uridine incorporation in sterile males carrying the balanced X-16 reciprocal translocation. The results failed to show an overall reactivation of the X as has been postulated by Lifschytz and Lindsley (1972) but there was some spreading of X inactivation along the translocated and normal chromosome 16 in those regions that were close to the X breakpoint. We feel that this process could be responsible for metabolic disturbances leading to degeneration of primary spermatocytes and, therefore, to sterility.