Structure of the major block of alphoid satellite DNA on the human Y chromosome.

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.

Extensive sequence homologies between Y and other human chromosomes.

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.

Y chromosomal DNA of Drosophila hydei.

Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome.

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.

Position effect variegation in Drosophila melanogaster: relationship between suppression effect and the amount of Y chromosome.

Position effect variegation results from chromosome rearrangements which translocate euchromatic genes close to the heterochromatin. The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing property of the D. melanogaster Y chromosome. We have determined the extent to which different cytologically characterized Y chromosome deficiencies and Y fragments suppress three V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes. We find that: (1) chromosomes which are cytologically different and yet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 60-80% of the entire Y, after which the effect reaches a plateau. These findings suggest suppression is a function of the amount of Y heterochromatin present in the genome and is not attributable to any discrete Y region.

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.

Y chromosome loops in Drosophila melanogaster.

Primary spermatocyte nuclei of fixed testes of Drosophila melanogaster exhibit three large clusters of thread-like structures, each consisting of two long, continuous, loop-shaped filaments. No comparable intranuclear structures are observed in spermatogonia, secondary spermatocytes or spermatids. The threads begin to form in young spermatocytes, grow throughout spermatocyte development, reach their maximum size in mature spermatocytes and disintegrate prior to meiotic metaphase I. The presence of each cluster of threads depends upon the presence of a specific region of the Y chromosome; when this region is deleted the cluster is absent, and when it is duplicated the cluster is also duplicated. Together these observations strongly suggest that these structures represent giant Y chromosome lampbrush-like loops analogous to those described in Drosophila hydei. Two antibodies, one polyclonal and one monoclonal, differentially react with the three loops of D. melanogaster. Moreover, two of these loops are specifically stained by Giemsa at pH 10. By indirect immunofluorescence with these antibodies followed by Giemsa staining, each loop can be unambiguously identified and its presence and normality readily assessed. This enabled us to perform fine mapping experiments to determine the relationships between the Y chromosome fertility factors and the loops. The loop-forming sites map within the kl-5, kl-3 and ks-1 fertility factors. Regions h3 and h21 of the Y chromosome correspond to the loop-forming sites of kl-5 and ks-1, respectively. Each of these regions contains about 1300 kb of DNA and spans about one-third of its locus. The loop-forming site of the kl-3 locus is coextensive with region h7-h9 which contains about 4300 kb of DNA and corresponds to the minimum physical size of this locus. These data suggest that each loop is an integral part of a different fertility factor, representing the cytological manifestation of its activity in primary spermatocytes. The kl-2, kl-1 and ks-2 fertility regions do not produce any visible intranuclear structure and do not affect the kl-5, kl-3 and ks-1 loops. Thus, these loci may either not form loops at all or produce loop-like structures that we are unable to see because they are physically minute, destroyed by our fixation procedure, or both.

Location and underreplication of satellite DNA in Drosophila melanogaster.

The two light nuclear satellites (PCsC1 = 1.672 and PCsC1 = 1.687) have been quantified in DNA isolated from the larvel imaginal discs and brains of Drosophila melanogaster with the genotypes X/O, X/X and X/Y. By comparing the results from these different genotypes, the amounts of the two satellites in the X and Y chromosomes and in the autosomes have been determined. The lightest satellite is not located to any appreciable extent in the X chromosome. The heterochromatic regions are not completely filled by these satellites. --Satellite DNA has also been quantified in DNA isolated from adults containing different genotypes. The two satellites are underreplicated to different extents. The apparent amount of underreplication for one of the satellites is different in different parts of the genome.

Effects of counterstaining with DNA binding drugs on fluorescent banding patterns of human and mammalian chromosomes.

Pairs of fluorescent A-T specific dyes and nonfluorescent agents with similar or complementary base pair binding specificity were used to analyse the extent to which banding patterns in human chromosomes obtained by fluorescent staining can be modified by counterstaining. By testing a variety of different combinations of drugs, essentially three types of alterations were observed. Enhanced contrast of specific heterochromatic regions was obtained with pentamidine, or netropsin, in conjunction with the fluorescent stains Hoechst 33258, DAPI or DIPI, the resulting banding patterns being similar to that reported for distamycin A plus DAPI (DA-DAPI banding [21]. Uniform quenching of Hoechst 33258, DAPI or DIPI fluorescence was induced by counterstaining with stilbamidine or berenil. The combination of echinomycin with DAPI resulted in an improved contrast of DAPI banding on chromosome arms and pale fluorescence on major autosomal C band regions. In addition, a subdivision of the heterochromatic part of the Y chromosome may be discerned by this latter technique.

Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification.

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.

Simultaneous application of immunolabelling and in situ hybridization to detect the origin of B and T lymphocytes in a case of acute lymphocytic leukaemia after bone marrow transplantation.

Previous cytogenetic studies, using selective mitogens, on a patient with B cell acute lymphocytic leukaemia during the 6 years of remission after bone marrow transplantation from an HLA-identical sister indicated persistence of recipient B lymphocytes in the peripheral blood. Such studies are necessarily limited to dividing cells at metaphase, which represent only a small proportion of the total cell population. We have now combined the techniques of immunolabelling and in situ hybridization on the patient's peripheral blood lymphocytes in order to define accurately their individual lineage and gender. A clear difference in the proportion of the persisting recipient lymphocytes was found between B and T lymphocyte lineages.

Chimerism studies using in situ hybridization for the Y chromosome after T cell-depleted bone marrow transplantation.

In situ hybridization for the Y chromosome (Y-ISH) was used to monitor engraftment in 10 patients with hematological malignancies who had received T cell-depleted marrow transplants from sex-mismatched donors, seven of whom were only partially HLA-matched. In the three patients who engrafted, as the peripheral counts rose, the percentage of host peripheral blood and marrow mononuclear cells decreased steadily, although host cells (less than 1%) could still be detected as late as day 252. The percentage of host granulocytes fell rapidly to less than 0.2%. Seven patients did not achieve full engraftment by day 28. Those with a low percentage of host cells (less than 1%) improved with observation or treatment with steroids, while those with a high or increasing percentage of host cells did not improve even after treatment with GM-CSF or with repeat marrow infusion without reconditioning. In one patient with graft failure, the residual host cells were predominantly CD8+ CD57+ and CD3+ CD56+, phenotypes consistent with non-MHC-restricted cytotoxic T cells. Lack of full engraftment in recipients of T cell-depleted marrow is not always associated with autologous reconstitution and does not always require retransplantation. Y-ISH may be useful for monitoring patients at high risk for graft failure in order to detect adverse trends in mixed chimerism that will alter therapy early after transplantation.

In situ hybridization in hematopathology.

In situ hybridization (ISH) is one of the molecular techniques that has applications in diagnostic hematopathology. This procedure allows the detection of DNA or RNA in intact cells from various preparations, including cytology specimens and routinely fixed paraffin-embedded tissues. ISH is therefore analogous to detecting proteins (antigens) in intact cells with immunohistochemistry. The purpose of this article is to review the basic concepts and principles of ISH and to briefly discuss the important technical details of this procedure. Examples of potential applications of ISH in hematopathology are then discussed, including detection of Epstein-Barr virus, Y chromosome, and oncogene activation.

Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells.

A fast and sensitive method for detecting biotinylated deoxyribonucleic acid (DNA) probes was used for sex determination of cells and tissues by in situ hybridisation of a probe "specific" for the Y chromosome (pHY 2.1). Within 24 hours this procedure visualizes the Y chromosome in fetal and adult cells and tissue, without background noise. This procedure should facilitate antenatal determination of sex on small numbers of uncultured cells. The sensitivity of the procedure also permits the chromosomal assignment of genes present in low copy number.

Isolation of Y-chromosomal repetitive DNA sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male DNA.

Male or female DNA of Drosophila hydei was sheared by sonication, denatured, reannealed to different C0t-values and fractionated by hydroxyapatite. The highly repetitive, moderately repetitive and unique fractions of female DNA were denatured again and coupled via diazotization or cyanogen bromide to macroporous Sephacryl S-500. Enrichment of Y-chromosomal sequences was achieved by cycling each of the different fractions of male DNA under optimized hybridization conditions over a column with a manifold excess of immobilized female DNA of the corresponding complexity. Thereby, Y-chromosomal sequences of D. hydei could be enriched about 100-fold for highly and moderately repetitive DNA and about 10-fold for unique DNA. When a library of male D. hydei DNA was screened with Y-enriched highly repetitive DNA, more than 98% of all hybridizing phages contained inserts of repetitive Y-chromosomal DNA of at least four different sequence families.

The effect of heating on Y-chromosome detection.

The effect of heating on Y-chromosome detection was investigated. Heat-treated blood was divided into four groups according to the hemolysis pattern obtained by the coil planet centrifuge system. A significant difference between the Y-positive nuclei of males and females was observed up to group III of the hemolysis pattern, and it was possible to determine the sex of the blood donor in spite of karyolysis and degeneration of blood components. However, sex determination of blood in group IV, indicating complete hemolysis, was impossible because of overlapping between male and female Y-chromosome counts. Practical application of this sex determination method was successful even with severely burned cadavers. However, it was suggested that putrefaction together with heat damage made the identification nearly impossible.

Variability of euchromatic and heterochromatic regions of Y chromosome in two types of cancer patients.

Heteromorphism of Y chromosome was studied in head and neck cancer patients and leukemia patients. The results were compared with similar data obtained for healthy men. It was observed that, compared to the controls, mean lengths of Y chromosome were nonsignificantly higher for leukemia patients and lower for head and neck cancer patients. The euchromatic region of Y chromosome (Y-eu) remained comparable in the controls and the leukemia patients, whereas it was smaller in patients with head and neck malignancies. The heterochromatic region (Y-het) was more or less analogous in controls and head and neck cancer patients, however, it was significantly larger in patients with leukemia (P less than 0.02).

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm.

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.

The determination of sex from forcibly removed hairs.

The determination of sex from forcibly removed hairs in forensic science laboratories has, in the past, been based almost entirely on the presence or absence of the Y chromosome in the cells of the hair root sheath. Since the human male genotype is XY and the female is XX, a technique was devised that permits root sheath cells to be stained sequentially for the Y and then the X chromosome using quinacrine mustard. Following staining, the Y and the X chromosome fluorescence were observed, at pH 5.5 and 3.0, respectively, by epifluorescence. The X and Y chromosome counts obtained for a single hair root specimen were reported as a Y-X (Y minus X) score. The results reported show that specimens from males gave positive Y-X score while specimens from females gave negative Y-X scores. Results of an age study and blind trials were also reported.

SIMREP: a program simulating differential DNA replication.

During endoreplication, different organisms in different taxa carry out DNA syntheses without nuclear division. The result of such endocycles is either a polyploid nucleus or a polytene architecture of the chromosomes. Since not all sequences of the genome may be reduplicated simultaneously and to the same extent, endocycles provide an opportunity for primary cell differentiation at the DNA level as a result of DNA amplification or underreplication. We have designed a numerical model which simulates differential endoreplications. The program SIMREP is written in TURBO PASCAL 4.0 and can be executed on a PC/XT/AT with MS-DOS greater than or equal to 2.0. It uses diploid DNA contents derived from meiotic or mitotic nuclei together with data on amounts of DNA present after a given number of endocycles. SIMREP can be applied to genomes containing arbitrary numbers of chromosomes (maximum N = 24) to model details of their replication behaviour. It is also useful in analysing differential replication of single genes. The application of SIMREP is illustrated with two examples. (1) Female and male specific types of underreplication were found in the chironomid midge Prodiamesa olivacea. The heterosomes which appear homomorphic in metaphases were identified by their differential polytenization. (2) The Y chromosome of Drosophila nasutoides was assessed to ascertain whether its replication is regulated in parallel with, or independently from the large chromosome pair 4.