Haploid Set Of Chromosomes Research Articles

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus.

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 microM A23187 for 5 min were treated with 10 microg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

Mechanism of malsegregations at meiosis: premature centromere separation and precocious division in female Chinese hamsters stimulated with gonadotropic hormones

ABSTRACT Using female Chinese hamsters stimulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), we investigated the influence of hormonal stimulation upon meiotic segregation in oocytes. In 1,576 oocytes ovulated spontaneously from 197 non-treated mature females, the number (percentage) of hyperhaploid oocytes with more than 12 (12–14) chromosomes was 16 (1.0%). These cells had no extra single chromatids, but all had extra chromosomes. Single chromatids were seen in 7 (0.4%) cells with a haploid chromosome set. On the other hand, a total of 1,329 and 1,198 second meiotic (MII) oocytes from 64 mature females and 61 immature females stimulated with PMSG and hCG, respectively, were subjected to chromosomal analysis. Single chromatids were seen in 34 (2.6%) and 62 (5.2%) of these oocytes, respectively. Since these chromatids were mostly paired and the sister chromatids existed near each other in many cells, they may have separated from some chromosomes of haploid cells. Compared with the non-treated females, the frequency of cells with single chromatids was significantly greater in oocytes from both mature and immature females stimulated with PMSG and hCG. The number (percentage) of hyperhaploid cells from mature and immature PMSG-hCG-stimulated females, respectively, was 15 (1.1%) and 14 (1.2%), which was not significantly greater than that in non-treated females. Most of these cells had extra whole chromosomes but one oocyte from mature females and one from immature females had an extra single chromatid. These findings indicate that such hormonal stimulation induces premature centromere separation in MII oocytes and precocious division at anaphase I, which can be assumed by the presence of MII cells with extra single chromatids. Considering that no or less hyperhaploid MII oocytes with an extra single chromatid were seen in oocytes from spontaneous ovulation and from artificial ovulation on hormonal stimulation, these findings suggest that the major mechanism of malsegregations at first meiotic (MI) division is not a precocious division but rather, errors such as nondisjunction of homologous chromosomes (dyads).

Sonographic, Cytogenetic and DNA Analysis in Four 69,XXX Fetuses Diagnosed in the Second Trimester

Objective: To describe the ultrasound findings and its relationship with the cytogenetic study and the origin of the extra haploid chromosome set in four 69,XXX cases. Methods: Four pregnant women were referred because of abnormal 2nd trimester ultrasound. Karytoypes, FISH and DNA analysis were performed. Results: All cases presented asymmetrical intrauterine growth retardation, marked oligohydramnios and placental alterations and showed a 69,XXX karyotype. In three cases, DNA analysis allowed to establish the origin of the extra haploid chromosome set. Conclusions: At least three fetuses had a maternal extra haploid chromosome set. Thus, it has been possible to establish the main ultrasonographic markers and to observe the survival of the fetus until the second trimester when they have a maternal origin.

Unusual triploid males in a microchromosome-carrying clone of the Amazon molly, Poecilia formosa

The Amazon molly, Poecilia formosa, is an all-female fish of hybrid origin which reproduces by gynogenesis, i.e. it depends on sperm of males of closely related species to trigger parthenogenetic development of the embryo. Therefore the offspring is clonal and identical to the mother. In rare cases the exclusion mechanism fails and paternal introgression occurs. This may result either in triploid offspring – if the whole haploid chromosome set of the sperm fuses with the diploid egg nucleus – or in siblings with microchromosomes – if only subgenomic amounts of paternal DNA are included. In one of our diploid, microchromosome-carrying laboratory stocks we observed eight triploid individuals which all developed into males. We investigated the mitotic and meiotic chromosomes, the synaptonemal complex (SC), and sperm production of these males, and compared them to males of the gonochoristic parental species (P. latipinna and P. mexicana) and their hybrids. This comparison revealed that P. formosa males are functional males with reduced effective fertility. They show a deviation from the typical 23 bivalents in the synaptonemal complexes as well as in diakinesis due to the triploid state. They produced offspring but only with gynogenetic Amazon molly females. This shows that the probably aneuploid sperm from P. formosa males can trigger parthenogenetic development of unreduced eggs.

Development of testes and differentiation of germ cells in water frogs of the Rana esculenta - complex (Amphibia, Anura)

Abstract The European water frog, Rana esculenta, is a hybrid whose genome is composed of haploid chromosome sets of its parental species R. lessonae and R. ridibunda. Prior to meiosis one of the parental sets is discarded and the other is duplicated (hybridogenesis). In the parental species sex differentiation begins at tadpole stages 28-30 (Gosner, 1960), at stages 30-36 the testes are composed of proliferating pale spermatogonia 1°. At stages 36-39 a new class of spermatogonia I° (dark) appears. Before first hibernation, seminiferous lobules are filled with cysts containing germ cells at various stages of spermatogenesis up to elongating spermatids. In R. esculenta gonad development is affected from the earliest stages: the gonads are smaller and composed of reduced number of spermatogonia I°. The phase of pale spermatogonia I° proliferation is prolonged up to the second year of life. The structure of the gonads, as well as that of germ cells themselves, are often abnormal.

The American Mink Gene Map

The American mink (Mustela vison) represents the large family Mustelidae. The family belongs to the order of Carnivora and includes many dozens of species living in a broad range of biotopes, from the Arctic to tropical zones. Carnivores are of great interest in comparative gene mapping in mammals. Giemsa banding with trypsin analysis has shown that chromosome divergence in Mustelidae, despite the considerable variation in number and morphology of chromosomes, mainly resulted from rearrangements of virtually constant elements, probably representing the remains of an ancestral karyotype (Graphodatsky and others 1989; Wuster-Hill and Centerwall 1982). A similar situation was reported in the families Canidae and Ursidae (Graphodatsky and others 1995; Nash and O'Brien 1987; Wurster-Hill and Centerwall 1982). Moreover, the comparison of the Giemsabanding chromosomes of Carnivora and species belonging to other orders—primates, rodents, or ungulates—demonstrated that in some cases there is a similarity in the Giemsabanding patterns for large chromosome regions of the distantly related species (Graphodatsky 1989; Nash and O'Brien 1982). However, supplementary data on gene mapping are needed to decide whether this similarity reflects true homology and to reach reliable conclusions about the chromosome evolution of mammals (O'Brien and others 1993; Wakefield and Graves 1996). For a long time, the efforts of mink breeders were focused on the identification of mutations affecting coat color genes. At the time of this writing, approximately 30 coat color mutations have been identified in mink. Most of them (at least 20) are controlled by independent autosomal loci (Nes and others 1988; Robinson 1975; Shackelford 1948). It is reasonable to expect that some of them are linked because a haploid set of mink chromosomes contains 14 autosomes plus the X or Y sex chromosome (Christensen and others 1996; Mandahl and Fredga 1975). However, there has been only 1 description of linkage of 2 coat color mutations, Ebony (Eb) and royal pastel (b) (Shackelford 1949). In this paper, I present the current state of gene mapping of mink, based on data obtained from traditional breeding, somatic cell hybridization, chromosome-mediated or interphase nuclei-mediated gene transfer procedures, and in situ hybridization.

Cytogenetic characterization of the genome of mealybugPlanococcus citri(Homoptera, Coccoidea)

Summary Mealybugs although being agriculturally harmful insects have been very poorly studied by modern cytogenetics techniques, and no cytotaxonomic criteria to distinguish between closely related species is available yet. In the mealybug Planococcus citri (2n=10) male and female individuals are both diploid, however in males, at the stage of blastula, the haploid chromosome set of paternal origin becomes heterochromatic, even though its complete inertia has been considered questionable. Here we present data on the cytogenetic characterization of the chromosomes of Planococcus citri. We report on (i) the fluorescence karyotype (D287/170), which to our knowledge is the first banded karyotype of a mealybug to be described; (ii) the chromosome localization of constitutive heterochromatin; (iii) the chromosome localization of rDNA sites; (iv) NORs activity. Our data also show, for the first time, that in the heterochromatic chromosome set ribosomal genes are still active.

Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization

A colored banding pattern for human chromosomes is described that distinguishes each chromosome in a single fluorescence in situ hybridization with a set of subregional DNA probes. Alu/polymerase chain reaction products of various human/rodent somatic cell hybrids (fragment hybrids) were pooled into two probe sets that were labeled differentially and detected by red and green fluorescence. Chromosome regions hybridized by DNA present in both pools appeared yellow. The result was a multi-color set of 110 distinct signals per haploid chromosome set for the human karyotype. Each individual chromosome showed a unique sequence of signals, a result termed the "chromosome bar code". The reproducibility of the hybridization pattern in various labeling and hybridization experiments was analyzed by computer densitometry. We have applied the chromosome bar code both in diagnostic cytogenetics and in genome studies. The approach allows the rapid identification of chromosomes and chromosome rearrangements. Although not yet showing the resolution of classical banding patterns, the present experiments demonstrate various applications in which the present multi-color bar code can significantly add to the spectrum of cytogenetic techniques.

Participation of the female pronucleus derived from the second polar body in full embryonic development of mice.

The second polar body (2PB), extruded from metaphase II oocytes after fertilization or oocyte activation, has a haploid set of female chromosomes like its sister, the fertilized (activated) oocyte. In the present study, the female pronucleus of fertilized mouse oocytes (zygotes) was replaced with the 2PB nucleus from the same or different oocytes to examine the developmental potential of the 2PB nucleus. When the female pronucleus (FPN) was synchronously (FPN and 2PB were same age) replaced with the 2PB nucleus, the rate of reconstructed zygotes developing to blastocysts decreased with the age of donors and recipients (from 70% at 20-21 h to 15% at 26-27 h after hCG injection). When nuclei were replaced asynchronously (FPN and 2PB were of different ages), a higher developmental rate to blastocysts was obtained with young recipient zygotes (20 h after hCG injection) than with aged recipient zygotes (24 h after hCG injection) (64% versus 20%, P < 0.01) irrespective of the age of the 2PB. In this second group of embryos, in which nuclei were replaced asynchronously, the 2PB nuclei were prematurely condensed at the time of first mitosis. These findings indicate that after being extruded from the oocytes the cell cycle of the 2PB progressed more slowly than did that of the zygote. After the transfer of reconstructed embryos into pseudopregnant females, normal pups with an expected coat colour were born, indicating the competence of the 2PB chromosomes for full embryo development.

Fluorescene in situ hybridization establishes homology between human and silvered leaf monkey chromosomes, reveals reciprocal translocations between chromosomes homologous to human Y/5, 1/9, and 6/16, and delineates an X1X2Y1Y2/X1X1X2X2 sex-chromosome system.

We employed in situ hybridization of chromosome-specific DNA probes ("chromosome painting") of all human chromosomes to establish homologies between the human and the silvered lead monkey karyotypes (Presbytis cristata 2n = 44). The 24 human paints gave 30 signals on the haploid female chromosome set and 34 signals on the haploid male chromosome set. This difference is due to a reciprocal translocation between the Y and an autosome homologous to human chromosome 5. This Y/autosome reciprocal translocation which is unique among catarrhine primates has produced a X1X2Y1Y2/X1X1X2X2 sex-chromosome system. Although most human syntenic groups have been maintained in the silvered leaf monkey chromosomes homologous to human chromosomes 14 and 15, 21 and 22 have experienced Robertsonian fusions. Further, the multiple FISH signals provided by libraries to human chromosomes 1/9, 6/16 indicate that these chromosomes have been split be reciprocal translocations. G-binding analysis shows three different forms of chromosome 1 (X2) which differ by a complex series of inversions in the 10 individuals karyotype. Comparisons with the hybridization patterns in hylobatids (gibbons and siamang) demonstrate that resemblances in chromosomal morphology and banding previously taken to indicate a special phylogenetic relationship between gibbons and colobines are due to convergence.

Structure and transcription of the gene for translation elongation factor 1 subunit alpha of zebrafish ( Danio rerio)

The zebrafish gene for translation elongation factor 1 α (EF1 α) was isolated from a phage Lambda genomic library and sequence and structure determined. One gene copy of EF1 α per haploid set of chromosomes was found and no processed pseudogenes. A highly active promoter region was localized to a 277 bp PstI/ PvuII fragment beginning 240 bp upstream from the tsp, but no transcription enhancing, or silencing activity was observed within 1 kbp upstream, or downstream from the promoter. Expression of EF1 α appears to be developmentally regulated.

Isolation and Characterization of the Human Chromosomal Gene for Prostacyclin-Stimulating Factor

Prostacyclin-stimulating factor (PSF) is a protein which acts on vascular endothelial cells and stimulates the production of prostacyclin. Recently, we were able to purify PSF from the conditioned medium of cultured human diploid fibroblasts and clone PSF cDNA. In this study, we screened a human genomic library and isolated genomic clones to determine the structure of the human chromosomal PSF gene. By determining the nucleotide sequence and transcription initiation site of this gene, we found that it comprises 5 exons and 4 introns. Southern hybridization analysis indicated the presence of a single copy of the PSF gene per haploid set of chromosomes. The 300 bp upstream of the transcription initiation site had a very high GC content, and 7 binding sites for the transcription regulating factor Sp1 were present.

Spermatocyte chromosomes and genome size in the sacoglossan slugStiliger felinus(Hutton 1882) (Gastropoda: Opisthobranchia) from New Zealand

Abstract A karyological investigation was carried out on the testis cells of the tidal pool sacoglossan sea slug Stiliger felinus from New Zealand. The haploid chromosome set was characterised by 14 elements where median‐, subterminal‐, and terminal‐centromere chromosomes were present. Using flow cytometric analysis, haploid genome size of the species was found to be 2.8 pg: the highest value encountered in the subclass Opisthobranchia. Stiliger felinus differs from other Stiligeridae in chromosome number and frequency of cross‐shaped bivalents in spermatocytal metaphase I plates.

Karyological variation in the group ofMyosotis alpestris (boraginacea)

A total of 6 population samples ofMyosotis stenophyllaKnaf, a rare species showing great ecological disjunction in its distribution, were examined to clarify the present status of its karyological variation. In order to elucidate relationships between lowland tetraploid populations ofM. stenophylla and diploid and tetraploid montane populations ofM. alpestrisF.W. Schmidt, four population samples ofM. alpestris were also examined. The karyotypes of all populations ofM. alpestris s.l. studied were highly asymmetrical and heterogeneous, being composed of metacentric, submetacentric, subtelocentric and satellited acrocentric chromosomes. The karyotype formula for haploid chromosome set was established: n=x=12=6m+2sm+3st+1tSAT. Multivariate analysis based on chromosome length and shape showed significant differences between diploid and tetraploid forms ofM. alpestris s.l. Four numerical parameters, used to characterize the karyotype ofM. stenophylla, revealed significant differences between populations on serpentine and on non-serpentine substrates. In addition, the noticeable affinity of the karyotype of non-serpentine populations to that ofM. alpestris tetraploids has been shown by means of discriminant analysis. These data suggest that the unique features of serpentine play an important role in the origin of karyotypic differentiation within populations ofM. stenophylla.

Effects of impaired insulin secretion on the fertilization of mouse oocytes.

We have studied the effect of moderately impaired maternal insulin secretion on oocyte chromosomal constitution, fertilization and zygote DNA synthesis. Female mice were injected with a single dose of streptozotocin (65 mg/kg) 14 days before fertilization/ovulation. Zygotes/oocytes were recovered from control and subdiabetic mice on day 1 of pregnancy. Compared with control animals, subdiabetic females showed a significant difference in the proportion of zygotes/oocytes. The subdiabetic mothers had a lower percentage of zygotes and a higher percentage of unfertilized and degenerated oocytes in comparison with control animals. The investigation of [3H]thymidine incorporation did not show any influence of the maternal subdiabetes on the initial zygote DNA synthesis. An analysis of the ovulated oocytes at the metaphase II stage isolated from subdiabetic mice did not reveal increased chromosomal anomalies in comparison with the controls. Control and subdiabetic mothers had a similar percentage of oocytes with a normal haploid set of chromosomes, and the incidence of aneuploidy/diploidy did not differ significantly. These observations suggest that insulin changes in subdiabetic mothers had a deleterious influence on oocyte fertilization in mice, but apparently they did not have any effect on the nuclear events.

Maternal origin of extra haploid set of chromosomes in third trimester triploid fetuses

Twenty-six highly polymorphic markers were used to determine the origin of the extra haploid chromosome set in 6 triploid fetuses of type II phenotype. All had reached the third trimester of pregnancy. The extra set was maternal in origin in all cases, supporting previous research indicating longer in utero survival of maternally-derived triploid fetuses. These findings provide evidence for an instance of genomic imprinting in humans.

Parental origin of the extra haploid chromosome set in triploidies diagnosed prenatally

The parental origin of the additional chromosome complement in a total of 17 cases of triploidy was determined mainly using highly polymorphic microsatellites. Maternal origin of the triploidy was demonstrated in most cases. To the best of our knowledge, this is the first systematic evaluation of the parental origin of chromosome sets in fetuses who survived until a cytogenetic diagnosis was established. In contrast to previous investigations this study documented a predominance of maternal origin of the extra haploid set mainly due to longer survival time for digynic triploidies. The concept of 2 distinct fetal phenotypes in triploidy is clearly supported by this study.

Evaluation of meiosis in cucumber (Cucumis sativusL.) monohaploids

SUMMARYDetailed meiotic studies were conducted for the first time on nine haploid plants representing four different genotypes of cucumber (Cucumis sativus L. n = x =7). At diakinesis and metaphase I frequency of rod type bivalents was very low (0.1 per pollen mother cell—PMC) being observed in 4.3% of PMCs of one haploid plant. The frequency of diads, triads and polyads (72–84%) was higher than tetrads and pollen was 100% sterile. Very rare bivalents associations and fragmentations of chromosomes are discussed as well as divisions of chromosomes up to chromonema. It is concluded that very little chromosome homology and no evidence of duplication is to be found within the haploid set of cucumber chromosomes and that the basic chromosome number is seven.

Pattern and Regulation of Acetyl-CoA Carboxylase Gene Expression1–3

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long chain fatty acids. There is a single copy of the gene for acetyl-CoA carboxylase per haploid chromosome set. The gene contains two promoters whose primary transcripts are differentially spliced resulting in multiple forms of acetyl-CoA carboxylase mRNA. These mRNA species are different in the 5'-untranslated region, but contain the same coding region. Generation of different forms of the mRNA is tissue specific and controlled by physiological conditions. Two promoters contain an extensive array of cis-elements that perceive changes in the cellular environment signalling repression and induction of long chain fatty acid synthesis. The ability of the gene to respond to various lipogenic signals and the presence of the same coding sequence in all acetyl-CoA carboxylase mRNA species suggest that the biosynthesis of fatty acids required for multiple functions in the cells is primarily regulated at the gene level.

Reproductive capacity of the nucleus of the male gamete after completion of meiosis.

Our purpose was to investigate the possibility of achieving fertilization and subsequent normal embryonic development by injecting round spermatid nuclei into rabbit oocytes. Two-to four-cell-stage embryos developed after round spermatid nuclear injections into rabbit ooplasma could further develop in vitro up to the expanding blastocyst stage or in vivo up to complete gestation. The current findings show that the haploid set of chromosomes of round spermatid can pair with the chromosomes of the ootid to participate in complete fertilization and subsequent embryonic and fetal development. In addition, we suggest that postmeiotic modifications of the round spermatid are not required for the pairing of male gamete chromosomes with those of the ootid.