The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.     

Barring gene expression after XIST: maintaining facultative heterochromatin on the inactive X

X chromosome inactivation refers to the developmentally regulated process of silencing gene expression from all but one X chromosome per cell in female mammals in order to equalize the levels of X chromosome derived gene expression between the sexes. While much attention has focused on the genetic and epigenetic events early in development that initiate the inactivation process, it is also important to understand the events that ensure maintenance of the inactive state through subsequent cell divisions. Gene silencing at the inactive X chromosome is irreversible in somatic cells and is achieved through the formation of facultative heterochromatin (visible as the Barr body) that is remarkably stable and faithfully preserved. Here we review the many features of inactive X chromatin in terminally differentiated cells and address the highly redundant mechanisms of maintaining the inactive X chromatin.     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

Evolution of karyotype in haploid cell lines of Drosophila melanogaster

Seven continuous cell lines have been established in vitro from lethal embryos produced by the female sterile mutant mh 1182 of Drosophila melanogaster. Six lines show haploid metaphases. Karyotype analysis revealed a high level of aneuploid cells with frequent chromosome fragments. In three lines, haploid cells were quickly overgrown by diploid cells. Two lines were more stable but the proportion of haploid cells decreased with time. One line was stable, showing 80-90% of haploid cells for over 1 000 cell generations. Stable haploid clones have been isolated from two lines. Crossing of mh 1182/mh 1182 females with males bearing a ring X chromosome shows that the haploid genome retained in the cells is of maternal origin and that the diploid cells derive from pre-existing haploid cells. The appearance of the diploid cells and the conditions of karyotypic stability are analysed.     

Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.     

Haploid fruiting in Cryptococcus neoformans is not mating type alpha-specific

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATalpha strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATalpha-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.     

Construction and characterization of a NotI linking library of human chromosome 21

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.     

Heterochromatin,gene position effect and gene silencing

Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.     

Hematopoietic precursor cells transiently reestablish permissiveness for X inactivation

Xist is the trigger for X inactivation in female mammals. The long noncoding Xist RNA localizes along one of the two female X chromosomes and initiates chromosome-wide silencing in the early embryo. In differentiated cells, Xist becomes dispensable for the maintenance of the inactive X, and its function for initiation of silencing is lost. How Xist mediates gene repression remains an open question. Here, we use an inducible Xist allele in adult mice to identify cells in which Xist can cause chromosome-wide silencing. We show that Xist has the ability to initiate silencing in immature hematopoietic precursor cells. In contrast, hematopoietic stem cells and mature blood cells are unable to initiate ectopic X inactivation. This indicates that pathways critical for silencing are transiently activated in hematopoietic differentiation. Xist-responsive cell types in normal female mice show a change of chromatin marks on the inactive X. However, dosage compensation is maintained throughout hematopoiesis. Therefore, Xist can initiate silencing in precursors with concomitant maintenance of dosage compensation. This suggests that Xist function is restricted in development by the limited activity of epigenetic pathways rather than by a change in the responsiveness of chromatin between embryonic and differentiated cell types.     

Large-scale population study of human cell lines indicates that dosage compensation is virtually complete

X chromosome inactivation in female mammals results in dosage compensation of X-linked gene products between the sexes. In humans there is evidence that a substantial proportion of genes escape from silencing. We have carried out a large-scale analysis of gene expression in lymphoblastoid cell lines from four human populations to determine the extent to which escape from X chromosome inactivation disrupts dosage compensation. We conclude that dosage compensation is virtually complete. Overall expression from the X chromosome is only slightly higher in females and can largely be accounted for by elevated female expression of approximately 5% of X-linked genes. We suggest that the potential contribution of escape from X chromosome inactivation to phenotypic differences between the sexes is more limited than previously believed.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

Stabilized non-complementing diploids (Ncd) from fused protoplast products of B. subtilis

Non-complementing diploids (Ncd) displaying the parental phenotype can be selected from polyethylene glycol (PEG)-treated fused polyauxotrophic protoplasts of Bacillus subtilis. These bacteria carry the two parental genomes, but only one of them is phenotypically expressed, the other being replicated but not expressed. Cellular cloning and DNA-DNA in situ hybridization led to the discovery of non-complementing diploid cells which at first sight could have been considered as parental haploids. The new class of stabilized Ncd (10(-7) segregants) can be obtained either directly after the primary fusion event or from segregating Ncd after further growth. The totally inactive chromosome of a stable Ncd can be activated after PEG-induced self fusion. DNA-mediated transformation studies using crude stable Ncd lysates as DNA donors show low frequencies for the genetic markers from the 'silent' chromosome. Contrary to the unstable Ncd situation, however, these frequencies remain low even with purified donor DNA. The differences in the transformation properties of the non-expressed markers are correlated to Ncd clone stability. These facts suggest that chromosome inactivation in PEG-induced fusion involves at least a two-stage process. The first would be reversible and the second irreversible, thus preserving the inactive chromosome state.     

Meiotic and Mitotic Behavior of Dicentric Chromosomes in SACCHAROMYCES CEREVISIAE

Meiotic recombination between a circular and a linear chromosome in Saccharomyces cerevisiae has been investigated. The circle was a haploid-viable derivative of chromosome III constructed by joining regions near the two chromosome ends via a recombinant DNA construction: (HMR/MAT-URA3-pBR322-MAT/HML) and was also deleted for MAL2 (which therefore uniquely marks a linear chromosome III). Recombination along chromosome III was measured for eight intervals spanning the entire length of the circular derivative. Only 25% of all tetrads from a ring/rod diploid contained four viable spores. These proved to be cases in which there was either no recombination along chromosome III or in which there were two-strand double crossovers or higher order crossovers that would not produce a dicentric chromosome.--At least half of the tetrads with three viable spores included one Ura+ Mal+ spore that was genetically highly unstable. The Ura+ Mal+ spore colonies gave rise to as many as seven genetically distinct, stable ("healed") derivatives, some of which had lost either URA3 or MAL2. Analysis of markers on chromosome III suggests that dicentric chromosomes frequently do not break during meiosis but are inherited intact into a haploid spore. In mitosis, however, the dicentric chromosome is frequently broken, giving rise to a variety of genetically distinct derivatives. We have also shown that dicentric ring chromosomes exhibit similar behavior: at least half the time they are not broken during meiosis but are broken and healed during mitosis.--The ring/rod diploid can also be used to determine the frequency of sister chromatid exchange (SCE) along an entire yeast ring chromosome. We estimate that an unequal number of SCE events occurs in approximately 15% of all cells undergoing meiosis. In contrast, the mitotic instability (and presumably SCE events) of a ring chromosome is low, occurring at a rate of about 1.2 X 10(-3) per cell division.     

Toward a long-range map of human chromosomal band 22q11

Human chromosome band 22q11 is involved in numerous chromosomal rearrangements. A long-range molecular map of this region would allow the more precise localization of the various breakpoints of these rearrangements. Toward this goal we have constructed a genomic DNA library that allows the isolation of DNA clones that are directly adjacent to NotI sites. NotI was chosen because it is a restriction enzyme that digests infrequently in the human genome. The genomic DNA used in this library was from a human/hamster hybrid cell line that has a chromosome 22 as the only visible human chromosome. Two clones were isolated and mapped to different regions of 22q11 using a somatic cell hybrid mapping panel. A long-range restriction map flanking the NotI site of each of these two clones was produced using NotI and other infrequently cutting enzymes. Both NotI sites analyzed were located in HTF islands, regions often associated with the 5' end of genes. Thus, the NotI map of 22q11 may also aid in the cloning of undiscovered genes, giving a starting point for the study of duplication/deficiency syndromes of the region.     

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.     

Ribosomal DNA magnification in Saccharomyces cerevisiae.

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.     

Study of RecA-independent homologous recombination and a chromosomal rearrangement in the Escherichia coli strain carrying an extended tandem duplication

A heterozygous tandem duplication in the Escherichia coli deo operon region deoA deoB::Tn5/deoC deoD thr::Tn9 with the total length approximately 150 kb, which was obtained in the conjugational mating in the HfrH strain, was examined. By means of digestion with the NotI enzyme, pulsed-field gel electrophoresis, and the conjugational transfer of the duplication in the F- strain, the chromosomal rearrangement, which occurred in the duplication region upon its stabilization in the bacterial genome, was studied. In a more stable strain, two new NotI sites were shown to appear in the chromosomal region located close to the duplication, which might have resulted from the transposition of the IS50 sequence from Tn5. The data were also obtained indicating the possibility of secondary transposition of the chromosomal segment between the two new NotI sites (approximately 30 kb) in the region located near the duplication. With the use of rec+ and recA strains, two types of haploid and diploid segregants generated by the duplication were studied: DeoD+ (the DeoD+ allele is not expressed in the original duplication due to the polar effect of the deoB::Tn5 insertion) and DeoC DeoD. The segregation of DeoD+ clones was shown to be RecA-dependent, whereas the DeoC DeoD segregants selected on the medium that contained thymine at a low concentration (i.e., under conditions of thymine starvation) appeared at a rather high frequency. However, the relative frequency of haploid clones, which have lost the duplication, strongly decreased in the recA genome among segregants of both types.     

Genetic recombination in mutants of the anthracycline-producer Streptomyces griseus

It was found that genetic recombination occurs if two marked strains of Streptomyces griseus (leukaemomycin-producing strains IMET JA 3933 and IMET JA 5142) are grown together in mixed cultures on semisolid media. The crossing techniques used and the method for carrying out selective analysis were essentially the same as those described by HOPWOOD (1967, 1972). The parent strains used for crosses were marked with single or double nutritional requirements and with mutations for drug resistance. The crosses are quite self-sterile, yielding only in one combination stable prototrophic recombinants at a low frequency (10(-5) to 10(-6)). The majority of recombinants behaved as stable haploid genotypes. A series of four-point crosses of different types of auxotrophs was carried out. The results of these experiments do not provide sufficient data for constructing a chromosome map, but provide basic information on the possibilities of genetic analysis of the production of anthracycline antibiotics. The majority of crosses performed were not fertile at 28 degrees C but, surprisingly, in some crosses carried out at 34 degrees C viable colonies were detected on minimal media at frequencies from 10(-3) to 10(-2).