Mice cloned from olfactory sensory neurons

Cloning by nuclear transplantation has been successfully carried out in various mammals, including mice. Until now mice have not been cloned from post-mitotic cells such as neurons. Here, we have generated fertile mouse clones derived by transferring the nuclei of post-mitotic, olfactory sensory neurons into oocytes. These results indicate that the genome of a post-mitotic, terminally differentiated neuron can re-enter the cell cycle and be reprogrammed to a state of totipotency after nuclear transfer. Moreover, the pattern of odorant receptor gene expression and the organization of odorant receptor genes in cloned mice was indistinguishable from wild-type animals, indicating that irreversible changes to the DNA of olfactory neurons do not accompany receptor gene choice.     

Genomic rearrangements correlated with antigenic variation in Trypanosoma brucei

The capacity of African trypanosomes to express sequentially a large repertoire of different surface antigens during an infection enables the parasite to evade the immune response of its host, and makes attempts to produce a vaccine against the disease difficult. It is evident that point mutations cannot account for antigen diversity. Variable antigens like immunoglobulins are derived from an extensive family of genes of which only one is expressed in a given cell. As somatic tic recombination is involved in the immunoglobulin gene system, this similarity prompted us to search for somatic rearrangements in trypanosome variable antigen genes. We have constructed a recombinant plasmid containing approximately half the DNA sequence coding for a Trypanosoma brucei variable antigen and hybridised the inserted sequences to various restriction enzyme digests of nuclear DNA from different trypanosome clones. Differences in the sizes of restriction tion fragments hybridising to the inserted variable antigen coding sequence show altered positions of enzyme sites relative to this sequence, indicating different arrangements of DNA sequences around this gene in different trypanosome clones.     

Functional V region formation during in vitro culture of a murine immature B precursor cell line

B lymphocytes originate from pluripotential haematopoietic stem cells and differentiate into immunoglobulin (Ig)-producing cells. B-cell lineage differentiation is accompanied by two types of immunoglobulin gene rearrangements--rearrangement of V, D and J gene segments to create a functional V gene and rearrangement of CH genes for heavy-chain switching. These results, however, have been obtained mainly by analysis of immunoglobulin gene organization of myeloma cells. Baltimore and his colleagues have established Abelson murine leukaemia virus (A-MuLV)-transformed cell lines and found a few lines capable of carrying out kappa-gene rearrangement or undergoing isotype switching during in vitro culture. To study early B-cell lineage differentiation events, we have now also established A-MuLV-transformed cell lines which are capable of differentiating from mu- to mu+ and of undergoing continuing rearrangement of heavy-chain genes in culture. Analysis of immunoglobulin gene organization of these transformed cells revealed that mu- cells have already undergone DNA rearrangements involving JH segments but an additional rearrangement of JH segments is required for initiation of mu-chain synthesis. Southern blot analysis of the DNA and two-dimensional gel electrophoresis of intracytoplasmic mu-chain show that mu-chain diversity with respect to antigen specificity may be generated during this second rearrangement process. As no rearrangement of light-chain genes takes place in these cells, this implies that light-chain gene rearrangement requires some further change, or a different enzyme.     

Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, slw3 and slw6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after activation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.     

RNA-mediated epigenetic programming of a genome-rearrangement pathway

Genome-wide DNA rearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.     

Aberrant rearrangement of the kappa light-chain locus involving the heavy-chain locus and chromosome 15 in a mouse plasmacytoma

The creation of a functional antibody gene requires the precise recombination of gene segments initially separated on the chromosome. Frequently errors occur in the process, resulting in the formation of a non-functional gene. The non-functional genes can be generated by incomplete rearrangements, frameshifts, or the use of pseudo V or J joining segments. It is likely that these aberrant rearrangements arise by the same mechanism as is used in generating functional genes, a process which we have suggested may involve unequal sister chromatid exchange. Aberrant rearrangements of immunoglobulin genes occur in normal lymphocytes and play a major part in allelic exclusion. However, it has recently been suggested that aberrant rearrangements involving immunoglobulin and non-immunoglobulin genes may be involved in tumorigenesis. This suggestion has been stimulated by the frequent occurrence of translocations involving chromosomes known to carry immunoglobulin genes in B-cell malignancies. The rearrangement of non-immunoglobulin DNA to the heavy-chain locus has recently been reported. Some aberrant rearrangements of the kappa locus appear to be due to rearrangements to sites that do not include the conventional sequence for V gene segment joining. Here we describe an aberrant kappa rearrangement that has led to the joining of DNA from chromosomes 15, 6 and 12, and so appears to be the result of chromosomal translocations or transpositions. As 15/6 or 15/12 translocations have frequently been found in mouse plasmacytomas (as have analogous translocations in human lymphocyte tumours) this aberrant kappa rearrangement may be unique to the plasmacytoma from which it was isolated.     

拟南芥雄性不育突变体opw(only pollen wall)候选突变基因的克隆

在T-DNA插入突变体Salk_059463株系的群体中,筛选到两株雄性不育突变体,对TDNA序列上的一对引物进行PCR鉴定,结果表明:其基因组中没有T-DNA插入.遗传分析表明这两株雄性不育突变体由同一单个隐性基因控制,引起不育的主要原因是从花药发育的第8期开始,小孢子细胞质内容物逐渐减少直至消失,到花药发育的第12期,药室内的小孢子只剩下一个花粉壁空壳,故该突变体命名为opw(only pollen wall).利用图位克隆的方法对OPW基因进行了定位,结果表明OPW基因位于第二条染色体上分子标记T28M21和T3G21之间的12 kb区间内,该区间内一共有21个基因注释.通过克隆区间内的基因并测序发现opw-1突变体基因组中At2g40140基因编码序列的外显子在第289和第290个碱基之间插入了一个A碱基,而opw-2突变体基因组中At2g40140基因编码序列的外显子在第412和第413个碱基之间插入了一个T碱基,造成的编码序列移码使第424至第426碱基成为终止密码子,故At2g40140是编码OPW的候选基因.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Human gamma-chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7

Three gene families that rearrange during the somatic development of T cells have been identified in the murine genome. Two of these gene families (alpha and beta) encode subunits of the antigen-specific T-cell receptor and are also present in the human genome. The third gene family, designated here as the gamma-chain gene family, is rearranged in murine cytolytic T cells but not in most helper T cells. Here we present evidence that the human genome also contains gamma-chain genes that undergo somatic rearrangement in leukaemia-derived T cells. Murine gamma-chain genes appear to be encoded in gene segments that are analogous to the immunoglobulin gene variable, constant and joining segments. There are two closely related constant-region gene segments in the human genome. One of the constant-region genes is deleted in all three T-cell leukaemias that we have studied. The two constant-region gamma-chain genes reside on the short arm of chromosome 7 (7p15); this region is involved in chromosomal rearrangements identified in T cells from individuals with the immunodeficiency syndrome ataxia telangiectasia and observed only rarely in routine cytogenetic analyses of normal individuals. This region is also a secondary site of beta-chain gene hybridization.     

A single micromanipulated stem cell gives rise to multiple T-cell receptor gene rearrangements in the thymus in vitro

The extensive range of specificities of T-cell receptors is generated, as for immunoglobulins, by rearrangement of genetic information. Much valuable information about rearrangement processes has been inferred by comparing DNA from (monoclonal) lymphoid lines with germ-line DNA and, for B cells, from rearrangements in some Abelson murine leukaemia virus-transformed cell lines. However, because it is difficult to isolate and grow precursor populations, it has not proved possible to study rearrangements occurring in normal untransformed cells in vitro. Here we show that a single T-cell precursor colonizing an alymphoid thymus lobe in organ culture can generate multiple receptor beta-chain gene rearrangements. These observations provide unequivocal evidence for the intra-thymic diversification of the T-cell repertoire. They also offer the possibility of investigating rearrangement and its control in the clonal progeny of a single normal T-cell precursor without the perturbations involved in the use of viral transformation or the production of T-cell hybridomas.     

Rearrangement of two distinct T-cell gamma-chain variable-region genes in human DNA

Selective cloning procedures for T-cell-specific complementary DNAs have revealed the existence of a gene designated gamma as well as the main antigen receptor alpha- and beta-chain genes. The gamma-chain genes undergo rearrangement during T-cell differentiation but the patterns and complexity of such rearrangements differ markedly in mouse and human. In mouse, a panel of cytotoxic T-lymphocyte clones exhibit the same rearrangement pattern with a gamma-chain gene probe and a set of three gamma-chain variable (V) genes have been identified in the DNA. Clonal diversity in mouse seems to be confined to V-J (joining) regions. In contrast, human T-cell lines exhibit diverse rearrangements suggestive of a family of differing V gamma genes variously rearranging to the two gamma-chain constant (C) region genes. Here we report the cloning of two very different V gamma genes rearranged to J segments upstream of the two human C gamma genes. Both V gamma genes are rearranged productively but nucleotide sequence comparison shows that they possess very little homology with each other. This shows that human T-cell V gamma genes exist which differ significantly from each other at the nucleotide level and that such diverse genes can be usefully rearranged in different T cells.     

Evolutionary transfer of the chloroplast tufA gene to the nucleus

Evolutionary gene transfer is a basic corollary of the now widely accepted endosymbiotic theory, which proposes that mitochondria and chloroplasts originated from once free-living eubacteria. The small organellar chromosomes are remnants of larger bacterial genomes, with most endosymbiont genes having been either transferred to the nucleus soon after endosymbiosis or lost entirely, with some being functionally replaced by pre-existing nuclear genes. Several lines of evidence indicate that relocation of some organelle genes could have been more recent. These include the abundance of non-functional organelle sequences of recent origin in nuclear DNA, successful artificial transfer of functional organelle genes to the nucleus, and several examples of recently lost organelle genes, although none of these is known to have been replaced by a nuclear homologue that is clearly of organellar ancestry. We present gene sequence and molecular phylogenetic evidence for the transfer of the chloroplast tufA gene to the nucleus in the green algal ancestor of land plants.     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

Progress of gene targeting in mouse

Gene targeting is a powerful approach of studying the gene function in vivo. Specific genetic modifications, including simple gene disruption, point mutations, large chromosomal deletions and rearrangements, targeted incorporation of foreign genes, could be introduced into the mouse genome by gene targeting. Recent studies make it possible to do the gene targeting with temporal and spatial control.     

The map-based sequence of the rice genome

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.