Conserved repetitive DNA sequences (Bkm) in normal equine males and sex-reversed females detected by in situ hybridization

In situ hybridization with a cloned banded krait sex-specific repetitive DNA probe (Bkm) indicates a high concentration of Bkm sequences on the horse Y chromosome in both normal XY males and XY sex-reversed females. Lesser, but still significant, concentrations of Bkm sequences were mapped to horse chromosomes 3, 4, and 30.     

Bkm sequences are polymorphic in humans and are clustered in pericentric regions of various acrocentric chromosomes including the Y

Summary Probes of uncloned Bkm satellite DNA and a Drosophila clone 2(8), consisting mainly of GATA repeasts related to a major sequence component in Bkm, have been used to probe Southern blots of human male and female DNAs obtained from a Caucasian and an Australian aboriginal population and to human chromosomes in situ. Hybridization was observed to a distinct and an indistint series of bands against a smeared background. The same distinct bands are identified in the DNA samples with both probes, but are most readily detected using the uncloned Bkm probe. Most restriction bands are common to both populations and some are polymorphic. However, certain bands appear to be characteristic of the Australian aboriginal samples. There are no distinct sex-linked patterns. However all of the small acrocentric human chromosomes, including the Y chromosome show hybridization to uncloned Bkm in situ.     

DNA fingerprinting analysis of vegetative compatibility groups in Aspergillus caelatus

Forty-three isolates of Aspergillus caelatus, whose vegetative compatibility groups (VCGs) have been identified, were assessed by DNA fingerprinting using a repetitive sequence DNA probe (pAF28) cloned from A. flavus. Thirteen distinct DNA fingerprint groups or genotypes were identified among the 43 isolates. Twenty-four isolates belonging to VCG 1 produced identical DNA fingerprints and included isolates from the United States and Japan. Four other DNA fingerprint groups had multiple isolates sharing identical fingerprints corresponding to VCGs 2, 3, 12 and 13. Eight of the 13 fingerprint groups corresponding to VCGs 4-11 were represented by a single isolate with a unique fingerprint pattern. These results provide further confirmation that the pAF28 probe can distinguish VCGs of species within Aspergillus section Flavi based on DNA fingerprint patterns and that the probe can be used to estimate the number of VCGs in a sample population. Most of the A. caelatus isolates produced fewer restriction fragments and weakly hybridized with the repetitive DNA probe pAF28 compared to hybridization patterns obtained with A. flavus, suggesting less homology of the probe to A. caelatus genomic DNA.     

Sex reversal in the mouse (Mus musculus) is caused by a recurrent nonreciprocal crossover involving the X and an aberrant Y chromosome

Satellite DNA (Bkm) from the W sex-determining chromosome of snakes, which is related to sequences on the mouse Y chromosome, has been used to analyze the DNA and chromosomes of sex-reversed (Sxr) XXSxr male mice. Such mice exhibit a male-specific Southern blot Bkm hybridization pattern, consistent with the presence of Y-chromosome DNA. In situ hybridization of Bkm to chromosomes of XXSxr mice shows an aberrant concentration of related sequences on the distal terminus of a large mouse chromosome. The XYSxr carrier male, however, shows a pair of small chromosomes, which are presumed to be aberrant Y derivatives. Meiosis in the XYSxr mouse involves transfer of chromatin rich in Bkm-related DNA from the Y-Y1 complex to the X distal terminus. We suggest that this event is responsible for the transmission of the Sxr trait.     

GATA repeats in the genome of Asellus aquaticus (Crustacea,Isopoda)

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A₃ staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic arcas of the male heterochromosome are not labelled.by E.R. Schmidt     

Sex-specific DNA in reptiles with temperature sex determination

Banded krait minor ("Bkm") satellite DNA, originating in the W-chromosome of the snake Bungarus fasciatus, has been found in the genome of diverse eukaryotic species including fruit fly, quail, and horse. Concentrations of Bkm have been found in the presumptive W-chromosome of snakes with isomorphic sex chromosomes and in the male-determining region of the Y-chromosome in mouse and man. We therefore asked whether Bkm-related DNA might be present in quantitative excess in DNA from males or females in two related species of sea turtle, Chelonia mydas, in which sex is determined by the temperature of the incubating egg, and Lepidochelys kempi, in which the critical sex-determining temperature has recently been described. Filter hybridization with the Bkm 2(8) probe revealed male-specific fragments in both species; female-specific fragments were also revealed in C. mydas. Sex-specific DNA sequences in temperature-sex-determined species such as Kemp's ridley and the green turtle were unexpected, but could be explained if there were an underlying genetic mode of sex determination in these animals, or alternatively, if temperature-influenced sex determination involved structural modifications in DNA adjacent to, or directly concerned with, the sex-determining genes. If these results are confirmed across a broader sample of sea turtles, the techniques described in this paper might be used routinely to identify gener in the young of these endangered animals, in which male and female are grossly indistinguishable.     

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.     

BKm minisatellite sequences are not sex associated but reveal DNA fingerprint polymorphisms in rainbow trout

GATA-GACA repetitive sequences first isolated from a female snake (termed BKm sequences) and associated with sex chromosomes in some species were hybridized to DNA from rainbow trout (Salmo gairdneri). Genomic DNA was studied from three groups of rainbow trout: (i) randomly selected males and females from an outbred group, (ii) androgenetic individuals from an inbred strain, and (iii) parents and offspring of an outbred strain. Three restriction enzymes (EcoRI, HaeIII, or HinfI) were used to digest the genomic DNA. The DNA was electrophoresed in agarose gels, transferred to nylon membranes, and the GATA-GACA repetitive sequence probe was hybridized to this DNA. There was no evidence of sex-associated patterns of hybridization with the enzymes used. However, the sequences reveal DNA fingerprint polymorphisms which appear to be inherited in a stable manner.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 x 10(-4) - 7 x 10(-6). The variability derived with the (TG)n probe in horses was higher than what we obtained with several other commonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)_n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 times 10-⁴-7 times 10-⁶ The variability derived with the (TG)_n, probe in horses was higher than what we obtained with several other commmonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

DNA fingerprints of Bombyx mori L. Testing of genotypic variability of parthenogenetic strains.

A comparative analysis of the total cellular DNA in certain parthenogenetic specimens of the silkworm (Bombyx mori), produced from the females of the parthenogenetic strains by two types of parthenogenesis, has been performed through the application of the DNA fingerprinting method based on M13 phage DNA as a hybridization probe. It has been shown that parental specimens and their genetically identical off-springs produced through ameiotic parthenogenesis have identical patterns of hybridization with the hypervariable DNA fragments. The off-springs produced through the meiotic type of parthenogenesis have individual-specific patterns of hybridization, revealing a high level of polymorphism of individual genotypes. The results obtained testify to the effectiveness and reliability of this promising method for identification of genotypic variability, marking and genomic characterization of parthenogenetic clones in the silkworm.     

Increased nondisjunction of chromosome 21 with age in human peripheral lymphocytes

Fluorescence in situ hybridization (FISH) on binucleated cells with chromosome-specific DNA probes provides a convenient way to visualize reciprocal segregation patterns in daughter nuclei, and overcomes most problems related to the artefactual loss or gain of chromosomes that flaw chromosome preparations. In this study, FISH was employed to evaluate age- and sex-effects on spontaneous malsegregation, nondisjunction and loss of chromosome 21 in human lymphocytes after the first division in culture. A total of 68 healthy nonsmokers and nondrinkers of alcohol (37 males and 31 females) were grouped by age as Group I (0-10 years), Group II (20-30 years), Group III (40-50 years) and Group IV (60-70 years), with at least seven subjects per group and sex. FISH with a pericentric chromosome 21 specific DNA probe was carried out on binucleated lymphocytes, cytokinesis-blocked by cytochalasin B (6 microg/ml for 26 h) at 44 h after initiation of cultures.Linear regression analyses demonstrated a significant age-related increase in the frequency of micronuclei without chromosome 21 (MN-21)(r=0.73, p<0.001 in females; r=0.69, p<0.001 in males) in all binucleated cells, with a steeper slope in females (0.1758) than in males (0. 1241). Analysis using the 2x2 chi-square (chi(2)) test on the frequencies of MN-21 showed significant age-related differences in both males and females, except males in Group III and Group IV (p>0. 05). A significant sex-related difference was found only in subjects over 60 years (p<0.05), with females having more MN-21 (12.57 per thousand vs. 8.43 per thousand) than males.Loss of chromosome 21, occurring at mean levels of 0.38 per thousand in all binucleated cells and 0.24 per thousand in binucleated cells containing four FISH signals, was shown not to be age- or sex-related. A positive age-related increase in nondisjunction of chromosome 21 was shown in males (r=0.50, p<0.01), females (r=0.61, p<0.001) and all subjects (r=0.55, p<0.001) by linear regression analysis. An age effect was found only between children and adults (p<0.01 for females, p<0.05     

Banded krait minor satellite (Bkm) contains sex and species-specific repetitive DNA

A novel class of repetitive DNA was isolated from a Bkm DNA library by exclusion hybridization. This sequence was mapped to the short arm of the W chromosome of banded krait, Bungarus fasciatus. Southern blot hybridization showed that these sequences are sex and species specific. Sequence analysis of a 206 bp long clone, BR87, revealed the presence of a tandem array of two internal repeat units of 18–19 bp alternating with each other with a gap of 1,2 or 3 nucleotides. To our knowledge, this is the first report of an exclusively W chromosome-and species-specific repeat isolated from any reptile. The functional significance of this sequence based on its organisation is discussed.     

Isolation and Characterization of a Mouse Y Chromosomal Repetitive Sequence

The Y chromosome plays a dominant role in mammalian sex determination, and characterization of this chromosome is essential to understand the mechanism responsible for testicular differentiation. Male mouse genomic DNA fragments, cloned into pBR322, were screened for the presence of Bkm (a female snake satellite DNA)-related sequences, and we obtained a clone (AC11) having a DNA fragment from the mouse Y chromosome. In addition to a Bkm-related sequence, this fragment contained a Y chromosomal repetitive sequence. DNA isolated from the XX sex-reversed male genome produced a hybridization pattern indistinguishable to that obtained with normal female DNA, suggesting that the AC11 sequence is not contained within the Y chromosomal DNA present in the sex-reversed male genome. Based on the hybridization patterns against mouse Y chromosomal DNA, AC11 classified 16 inbred laboratory strains into two categories; those with the Mus musculus musculus type Y chromosome and those with the M.m. domesticus type Y chromosome. Three European subspecies of Mus musculus (M.m. brevirostris, M.m. poschiavinus and M.m. praetextus) possessed the M.m. domesticus type Y chromosome, whereas the Japanese mouse, M.m. molossinus, had the M.m. musculus type Y chromosome. The survey was also extended to six other species that belong to the genus Mus, of which M. spretus and M. hortulamus showed significant amounts of AC11-related sequences in their Y chromosomes. The male-specific accumulation of AC11-related sequences was not found in M. caroli, M. cookii, M. pahari or M. platythrix. This marked difference among Mus species indicates that the amplification of AC11-related sequences in the mouse Y chromosome was a recent evolutionary event.     

A Bkm-associated human y-chromosomal DNA is conserved and transcribed in the testis of mouse

Y chromosome associated genes and repetitive sequences are continually viewed from the point of view of their possible involvement in sex determination and in the evolution of such a mechanism, thus sustaining an interest in the identification of novel sequences to gain newer insights. Here we have used the highly conserved class of Bkm repeats to isolate its associated sequences from the Y chromosome under the assumption that these sequences could be involved in sex determination and might also reflect the evolutionary status of the Y chromosome. Towards this end we have screened a genomic library enriched with human Y chromosome DNA with Bkm. One of the positive clones, C65, has a pericentromeric location on the Y chromosome and is present in a number of human sex-reversed XX males. The 10.5 kb insert of clone C65 has been further subcloned (pFI, pFII, pFIII, pFIV). The subclone pFIII is present in both sexes in human and mouse, whereas pFIV is primate specific and present in both sexes. pFII contains sequences homologous to Bkm. pFI is conserved in mouse and man, but is Y specific only in primates. Although present in both sexes in mouse, pFI is transcribed specifically in the male testis suggesting that it may be involved in the process of sex determination or testis differentiation and spermatogenesis.     

Features of the DNA fingerprinting probe pITZ1

Stringently controlled plasmids generate DNA fingerprint patterns in mammals when used at low hybridization temperatures. In order to develop a probe for use in paternity testing in cattle we screened a bovine, partial genomic plasmid library with the PCR-amplified ori region of plasmid P1. Of eight isolated clones one generated strong band patterns at high stringency in various mammalian species (data not shown). Sequence analysis revealed an imperfect, compound dinucleotide repeat region, which was PCR-amplified and cloned into the plasmid vector pUC19. Fingerprint results generated by this probe (termed pITZ1) in cattle are compared with the results generated by VNTR-probe pV47, which itself was developed by screening a human chromosome 16 library with tandem repeats of bacteriophage M13. Probe pITZ1 is useful in conjunction with other VNTR-probes for DNA fingerprinting in cattle and donkey populations. The dinucleotide repeat region responsible for the band patterns generated with pITZ1 is close to an Alu -like sequence, which may be involved in eukaryotic replication mechanisms.     

The sex determining region of Chironomus thummi is associated with highly repetitive DNA and transposable elements

The dominant male sex determiner in chromosome III of the midge Chironomus thummi thummi is closely linked to a large cluster of tandem-repetitive DNA elements, the Cla elements, which are otherwise highly repetitive and distributed over more than 200 sites on all chromosomes. Chromosome III displays a hemizygous cluster of Cla elements in males but not in females. The chromosomal location of this hemizygous Cla element cluster is in the region of the male determiner M as localized by cytogenetic analysis. With Cla elements as hybridization probe, it was possible to clone a large part of the sex determining region. Molecular analysis of the DNA of males and females in this region displayed a number of differences between the two sexes. One striking difference is an unusual transposable element associated with the male sex determining region. The sex determining region also contains several other tandem-repetitive DNA elements in addition to the Cla elements. They are interspersed with single copy DNA. The accumulation of repetitive elements in the sex determining region is interpreted as the result of a lack of recombination between the male/female heteromorphic region, although recombination in the other sections of chromosome III occurs.     

Sexually dimorphic expression of pituitary glycoprotein hormones in a sex-changing fish (Pseudolabrus sieboldi)

It is widely accepted that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in socially controlled sex-changing fish. However, the specific secretion profiles of pituitary gonadotropins (GtHs) in this type of fish are not known. To address this fundamental question, we demonstrated that the diurnal secretion patterns of GtHs differ distinctly between males and females in a socially controlled sex-changing fish. We analyzed the pituitary mRNA levels of glycoprotein hormone subunits (i.e., the common alpha-subunit and specific beta-subunits follicle-stimulating hormone beta, luteinizing hormone beta, and thyroid-stimulating hormone beta) in the wrasse Pseudolabrus sieboldi, which is a model fish that exhibits accurate diurnal rhythms of gametogenesis in both males and females. Northern blots clearly showed that each subunit gene exhibits a diurnal rhythm of expression in the pituitary and that the expression patterns differ distinctly between the sexes. Our results suggest that oogenesis and spermatogenesis in this hermaphroditic fish are regulated differentially through the distinct secretion patterns of pituitary glycoprotein hormones. This study also provides direct evidence of the sexual plasticity of pituitary GtH secretion in a socially controlled sex-changing fish.     

Satellite DNAs,Heterochromatin and sex chromosomes in Chionodraco hamatus (Channichthyidae,Perciformes)

We studied the genome of an antarctic ice fish, Chionodraco hamatus, in order to detect highly repetitive DNAs that may play a role in heterochromatinization processes and sex chromosome differentiation. We used two different experimental approaches. Hybridization of a Bkm probe to genomic DNA showed slight differences between the two sexes. Using restriction enzymes, a Bgl II satellite (pIF) was isolated. In situ hybridization revealed a preferential localization of pIF on the centromeres and the telomeres of most chromosomes, as well as an interstitial band on the long arms of the neo-Y sex chromosome, where probably the hypothetical fusion took place. Dot-blot experiments showed that pIF is still present in species belonging to different families of the same suborder. Though preliminary, our results suggest a conservative nature of this DNA which might have played a definite functional role in the genome of these polar fishes.