Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585–586 from a tail-to-head arrangement to a head-to-tail configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of satellite DNA and is thus designated X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either X (2D12/E2) or 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.     

Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.     

Isolation and characterization of an α-satellite repeated sequence from human chromosome 22

We constructed a library in IL47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag⁺/Cs₂SO₄ gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of -R1-DNA and the X specific -satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived -satellite sequences analogous to other chromosome-specific satellite sequences described previously.     

Conservation and chromosomal localization of DNA satellites in balenopterid whales

DNA satellites were isolated from three balenopterid species, viz. the minke, sei, and fin whales. In each of them at least two DNA satellites were recognizable with buoyant densities in neutral CsCl of =1.702/1.703 and =1.710/1.711, respectively. cRNAs from each satellite group were used for filter and in situ hybridisations. Homo- and heterologous DNA-cRNA hybrids within each satellite group yielded virtually identical melting curve profiles showing conservation of at least a considerable part of the DNA satellite sequences. There was no evident sequence homology between the =1.702/1.703 and the =1.710/1.711 satellites by filter hybridisation. — The in situ hybridisation showed that in each species the =1.702/1.703 satellite was located in centromeric-paracentromeric C-bands in a few pairs, whereas the =1.710/1.711 satellite was located in terminal C-bands throughout the karyotypes. — The data on the whale DNA satellites indicate that the quantitative evolution of the satellite DNA sequences preceded species divergence of the balenopterids and that the satellite sequences have remained relatively unaltered since the divergence took place. The function of satellite DNA is considered to imply the introduction of both chromosomal and genic polymorphisms and thus being of great importance in speciation. Based upon these concepts a model is postulated for the function of satellite DNA. According to this model at meiotic pairing euchromatin-heterochromatin overlapping between homologous chromosomes is considered to be of a general occurrence. This overlapping is presumed to be accentuated by the size heteromorphism frequently observed between homologous heterochromatic segments (C-bands). In the region of such euchromatin-heterochromatin overlapping, crossing-over would be excluded. The overlapping is suggested to be rectified progressively in the chromosome arms, leaving unaffected crossing-over distant to the euchromatin-heterochromatin junctions. The consequence of this will be that genes in the proximity of the junctions are collectively inherited and selected, whereas genes distant to the heterochromatin will be independently assorted and selected.     

Location of satellite DNAs in the chromosomes of the kangaroo rat (Dipodomys ordii)

The location of satellite DNA sequences in metaphase chromosomes has been studied in the kangaroo rat by the in situ hybridization technique, staining techniques and phase contrast microscopy. The HS- satellite DNA is located at the kinetochores of all but three chromosome pairs. The HD satellite is located predominantly in the short arms of the chromosomes containing HS- and in the kinetochores of chromosome pairs that lack HS-. The regions that contain the satellite DNA sequences can also be identified by the Giemsa staining technique, and can be visualized with phase contrast microscopy or following Feulgen staining of fixed chromosome preparations.     

Callus formation from the suspensor ofPhaseolus coccineus in hormone-free medium: a cytological and DNA cytophotometric study

Summary Intact embryos (with suspensor) ofPhaseolus coccineus (2 n=2 x=22) in different stages of development were grownin vitro on hormone-free medium under conditions favoring callus formation from the portion of the suspensor proximal to the embryo (handle portion). It was shown that callus formation was dependent upon the developmental stage of the embryo and the integrity of the embryo-suspensor system.Callus formation from the handle portion of the suspensor, whose cells have nuclei with DNA values from 4 C to 128 C, was initiated by amitosis (nuclear fragmentation) which led to binucleate and multinucleate cells. Amitosis was followed by mitosis of both nuclei resulting from a previous amitosis and nuclei of euploid cells (mostly diploid) pre-existing in the suspensor. The majority of mitoses (88%) had either a reduced chromosome number (hypodiploid, haploid, and hypohaploid) or the diploid number (22 chromosomes).The very high incidence of cells with reduced chromosome number in the suspensor callus is discussed in relation to its mechanism of origin and to its possible exploitation in plant regeneration experiments.     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

Repetitive DNA sequences in Drosophila

The satellite DNAs of Drosophila melanogaster and D. virilis have been examined by isopycnic centrifugation, thermal denaturation, and in situ molecular hybridization. The satellites melt over a narrow temperature range, reassociate rapidly after denaturation, and separate into strands of differing buoyant density in alkaline CsCl. In D. virilis and D. melanogaster the satellites constitute respectively 41% and 8% of the DNA isolated from diploid tissue. The satellites make up only a minute fraction of the DNA isolated from polytene tissue. Complementary RNA synthesized in vitro from the largest satellite of D. virilis hybridized to the centromeric heterochromatin of mitotic chromosomes, although binding to the Y chromosome was low. The same cRNA hybridized primarily to the -heterochromatin in the chromocenter of salivary gland nuclei. The level of hybridization in diploid and polytene nuclei was similar, despite the great difference in total DNA content. The centrifugation and hybridization data imply that the -heterochromatin either does not replicate or replicates only slightly during polytenization. Similar but less extensive data are presented for D. melanogaster. — In D. melanogaster cRNA synthesized from total DNA hybridized to the entire chromocenter (- and -heterochromatin) and less intensely to many bands on the chromosome arms. The X chromosome was more heavily labeled than the autosomes. In D. virilis the X chromosome showed a similar preferential binding of cRNA copied from main peak sequences.—It is concluded that the majority of repetitive sequences in D. virilis and D. melanogaster are located in the - and -heterochromatin. Repetitive sequences constitute only a small percentage of the euchromatin, but they are widely distributed in the chromosomes. During polytenization the -heterochromatin probably does not replicate, but some or all of the repetitive sequences in the -heterochromatin and the euchromatin do replicate.     

The beta chorionic gonadotropin-beta luteinizing gene cluster maps to human chromosome 19

Summary We used a cloned human cDNA probe homologous to the placenta chorionic gonadotropin subunit (CGB) and to the pituitary luteinizing hormone subunit (LHB) and Southern blotting techniques to analyse DNA from a series of rodent x human somatic cell hybrids for the presence of specific gonadotropin subunit related sequences. Our results provide evidence for the assignment and linkage of the eight genes (or pseudogenes) coding for the subunit of these glycoprotein hormones to chromosome 19. Moreover, we observed a strict concordance between the permissivity of mouse x man hybrid cells to enteroviruses (which is linked to the presence of specific cell receptors encoded by human chromosome 19) and the presence of CGB and LHB related sequences, thus confirming the localization of the structural genes for the subunits on chromosome 19.This work was supported in part by INSERM grants CRL 81 1041 and by MRC grant MT 4860     

The distribution of sequences complementary to human satellite DNAs I,II and IV in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Human satellite DNAs I, II and IV were transcribed to yield radioactive complementary RNAs (cRNAs). These cRNAs were hybridised to metaphase chromosomes of man, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus). The results of this in situ hybridisation were analysed quantitatively and compared with accepted chromosome homologies based on Giemsa banding patterns. The cRNA to satellite II (cRNAII) did not hybridise to chimpanzee chromosomes, although its hybridisation to chromosomes of gorilla and orang utan yielded more autoradiograph grains than hybridisation to human chromosomes, and cRNAIV hybridised to many chromosomes of gorilla and chimpanzee but was almost entirely restricted to the Y chromosome in orang utan. Most sites of hybridisation were located on homologous chromosomes in all four species, but there were a number of sites which showed no correspondence between satellite DNA location and chromosome banding patterns, and others where a given chromosomal location hybridised with different cRNAs in each species. These results are in contrast to those found for many transcribed DNA sequences, where the same sequence is usually located at homologous chromosome sites in different species, and appear to cast doubt on many proposed models of satellite DNA function.     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Notes on chromosome behaviour in the spermatogenesis of the damselflyEnallagma cyathigerum (Charp.) (Odonata: Coenagrionidae)

The chromosome number in the damselflyEnallagma cyathigerum (Charp.) is 2n =27, 2n =28, n =14 (13). The sex determining mechanism is XX-XO, the X segregates postreductionally in the male.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

A comparative chromosome study of twenty killifish species of the genus Fundulus (Teleostei: Cyprinodontidae)

Female karyotypes from ovarian cell cultures of 20 species of killifish (Fundulus) ranged in diploid number from 32 to 48, but in arm number (NF) from 48 to 52. The small F chromosomes, which constituted the fundamental elements in the karyotype, were evenly graded in length. The large biarmed chromosomes (L), which were about twice the length of the average Fs, characterized only those species with 2N less than 48 chromosomes. And among these species, an increase in complement by a pair of L's was always accompanied by a decrease of two pairs of A's, indicating Robertsonian changes by the centric fusion of two A's to form one L chromosome. Other diagnostic chromosome characters included: the number and structure of biarmed and satellited F chromosomes and the percentage of F's with relatively short short-arms (SSA). Besides centric fusion, mechanisms of chromosomal evolution in Fundulus probably included pericentric inversion, producing biarmed F chromosomes from acrocentric F's and partial loss of a chromosome segment producing smaller biarmed F chromosomes from larger ones. The percentage of SSA chromosomes generally decreases from relatively primitive to specialized species. The presumably most primitive species have only SSA type acrocentric F chromosomes. The 20 Fundulus species were classified into 2 major groups according to the percentage of SSA chromosomes: the SSA group, including 3 subgroups, had more than 50% SSA's; the LSA group, including 2 subgroups, had fewer than 50% SSA's. This classification based only on karyotypic characters generally agreed with others based on gross morphological characters. A possible evolutionary scheme is proposed to account for the derived killifish karyotypes.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Transposition of mobile genetic elements in interspecific hybrids of Drosophila

In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using ³²P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions (jumping) of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with synthetic karyotoypes characterized by different combinations of D. virilis homologous chromosomes and hybrid chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition (jumping) took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such synthetic stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.     

Human β-glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids

-Glucuronidase (GUS) has become an important enzyme model for the genetic study of molecular disease, enzyme realization, and therapy, and for the biogenesis and function of the lysosome and lysosomal enzymes. The genetics of human -glucuronidase was investigated utilizing 188 primary man-mouse and man-Chinese hamster somatic cell hybrids segregating human chromosomes. Cell hybrids were derived from 16 different fusion experiments involving cells from ten different and unrelated individuals and six different rodent cell lines. The genetic relationship of GUS to 28 enzyme markers representing 19 linkage groups was determined, and chromosome studies on selected cell hybrids were performed. The evidence indicates that the -glucuronidase gene is assigned to chromosome 7 in man. Comparative linkage data in man and mouse indicate that the structural gene GUS is located in a region on chromosome 7 that has remained conserved during evolution. Involvement of other chromosomes whose genes may be important in the final expression of GUS was not observed. A tetrameric structure of human -glucuronidase was demonstrated by the formation of three heteropolymers migrating between the human and mouse molecular forms in chromosome 7 positive cell hybrids. Linkage of GUS to other lysosomal enzyme genes was investigated. -Hexosaminidase HEX _B) was assigned to chromosome 5; acid phosphatase₂ (ACP ₂) and esterase A₄ (ES-A ₄) were assigned to chromosome 11; HEX _A was not linked to GUS; and -galactosidase (-GAL) was localized on the X chromosome. These assignments are consistent with previous reports. Evidence was not obtained for a cluster of lysosomal enzyme structural genes. In demonstrating that GUS was not assigned to chromosome 9 utilizing an X/9 translocation segregating in cell hybrids, the gene coding for human adenylate kinase₁ was confirmed to be located on chromosome 9.Supported by NIH Grants HD 05196, GM 20454, and GM 06321, by NSF Grant BMS 73-07072, and by HEW Maternal and Child Health Service, Project 417.     

Characterisation of a human Y chromosome repeated sequence and related sequences in higher primates

The human Y chromosome carries 2000 copies of a tandemly repeated sequence, 2.47 kb long, which constitutes about 20% of the DNA of this chromosome. These sequences are localised on the tip of the long arm of the Y chromosome. Related sequences are present in DNA of females with a related but distinguishable restriction pattern. These autosomal sequences are distributed in tandem arrays on a number of autosomes. Related sequences are also present in gorilla and chimpanzee. In gorilla they resemble the human sequences in their restriction map but are not found on the Y chromosome whereas in chimpanzee the related sequences behave as a dispersed repeat. Changes in the level of methylation of this sequence in different tissues of human males can be detected with the lowest levels found in sperm and placental DNA.     

Polytene chromosomes of Simulium craigi (Diptera: Simuliidae)

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype CC from Pennsylvania; cytotype AF from Ontario and Manitoba; and cytotype ACF/BCF from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.