Molecular characterization and cytological mapping of a non-repetitive DNA sequence region from the W chromosome of chicken and its use as a universal probe for sexing Carinatae birds

A non-repetitive genomic DNA region of about 25 kb was cloned from the W chromosome of chicken using a genomic library prepared from a single W chromosome of the chicken. This region was mapped by fluorescence in situ hybridization (FISH) with mitotic and lampbrush chromosomes to a position between the major EcoRI family and the pericentromeric XhoI family on the W chromosome. A 0.6-kb EcoRI fragment(EE0.6) subcloned from this region consists of a sequence that can be obtained by the exon-trapping procedure and flanking sequences. Sequences, which are closely similar to that of EE0.6, are widely conserved on the W chromosomes of Carinatae birds, as revealed by Southern blot hybridization to HindIII-digested female and male genomic DNAs from 18 species of birds belonging to eight different taxonomic orders. The female sex of those birds can be determined by the presence of an unambiguous female-specific band. For many species of birds, the female sex can also be determined by polymerase chain reaction (PCR) using a set of primers from the flanking sequences in the chicken EE0.6.This revised version was published online in November 2005 with corrections to the Cover Date.     

Molecular and cytological characterization of SspI-family repetitive sequence on the chicken W chromosome

A genomic clone, pWS44, isolated from the chicken W chromosome-specific genomic library contained a partial (226-bp) sequence of a novel SspI-family repetitive sequence. A genomic clone, pWPRS09, containing a 508-bp SspI fragment (a repeating unit of the family) was subsequently obtained and sequenced. This 0.5-kb unit is tandemly repeated about 11 300 times. FISH to mitotic and lampbrush W chromosomes indicates that the SspI-family is located on the chromomere 6 between heterochromatic and distal non-heterochromatic regions on the short arm. The SspI-family sequence was proved to be a good positional marker in FISH mapping of active genes in the non-heterochromatic region on the lampbrush W chromosome. The presence of SspI-family repetitive sequence is limited to the genus Gallus (chickens and jungle fowls). The 0.5-kb repeating unit contains a 120-bp stretch of polypurine/polypyrimidine sequence (GGAGA repeats), shows no DNA curvature, and rapid electrophoretic mobility in 4% polyacrylamide gel at 4°C. The SspI-family forms a relatively diffused chromatin structure in nuclei. These features are distinctly different from those of XhoI- and EcoRI-family sequences on the W chromosome. The total amount of non-repetitive DNA in the chicken W chromosome is estimated to be about 10 Mb. This revised version was published online in July 2006 with corrections to the Cover Date.     

Methylation of a euchromatin-heterochromatin transition region in Arabidopsis thaliana chromosome 5 left arm

Cytosine methylation was studied at the level of the euchromatin/heterochromatin transition genomic region of the Arabidopsis chromosome 5 left arm. It has been shown using a monoclonal antibody against 5-methylcytosines that the density of DNA methylation increases from the euchromatin towards the heterochromatin. YACs mapped along this region were characterized for their repeated sequences content. Some of them, corresponding to euchromatin, euchromatin/heterochromatin border and heterochromatin regions, were used as probes for a Southern blot analysis of methylation. This revealed that the degree of mCmCGG and GATmC methylation increases significantly from the euchromatin towards the heterochromatin. Moreover, an analysis of cytosine methylation levels (% of 5-methylcytosine) of different DNA fragments, inside the same genomic region, was performed using PCR and/or Southern blot approaches. There is a gradual increase of methylation along the genomic region analyzed: CpG methylation in the euchromatic fraction, CpG and CpNpG methylation at the euchromatin/heterochromatin transition and an additional asymmetrical methylation in the repeated-heterochromatic fraction. The most methylated repeated family at CpG, CpNpG and asymmetrical sites is the 5S ribosomal DNA, highly methylated even though it is transcribed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel family of repetitive DNA sequences amplified site-specifically on the W chromosomes in Neognathous birds

A novel family of repetitive DNA sequences was molecularly cloned from ApaI-digested genomic DNA of two Galliformes species, Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris), and characterized by chromosome in-situ hybridization and filter hybridization. Both the repeated sequence elements produced intensely painted signals on the W chromosomes, whereas they weakly hybridized to whole chromosomal regions as interspersed-type repetitive sequences. The repeated elements of the two species had high similarity of nucleotide sequences, and cross-hybridized to chromosomes of two other Galliformes species, chicken (Gallus gallus) and blue-breasted quail (Coturnix chinensis). The nucleotide sequences were conserved in three other orders of Neognathous birds, the Strigiformes, Gruiformes and Falconiformes, but not in Palaeognathous birds, the Struthioniformes and Tinamiformes, indicating that the repeated sequence elements were amplified on the W chromosomes in the lineage of Neognathous birds after the common ancestor diverged into the Palaeognathae and Neognathae. They are components of the W heterochromatin in Neognathous birds, and a good molecular cytogenetic marker for estimating the phylogenetic relationships and for clarifying the origin of the sex chromosome heterochromatin and the process of sex chromosome differentiation in birds.     

The role of LINEs and CpG islands in dosage compensation on the chicken Z chromosome

Most avian Z genes are expressed more highly in ZZ males than ZW females, suggesting that chromosome-wide mechanisms of dosage compensation have not evolved. Nevertheless, a small percentage of Z genes are expressed at similar levels in males and females, an indication that a yet unidentified mechanism compensates for the sex difference in copy number. Primary DNA sequences are thought to have a role in determining chromosome gene inactivation status on the mammalian X chromosome. However, it is currently unknown whether primary DNA sequences also mediate chicken Z gene compensation status. Using a combination of chicken DNA sequences and Z gene compensation profiles of 310 genes, we explored the relationship between Z gene compensation status and primary DNA sequence features. Statistical analysis of different Z chromosomal features revealed that long interspersed nuclear elements (LINEs) and CpG islands are enriched on the Z chromosome compared with 329 other DNA features. Linear support vector machine (SVM) classifiers, using primary DNA sequences, correctly predict the Z compensation status for >60% of all Z-linked genes. CpG islands appear to be the most accurate classifier and alone can correctly predict compensation of 63% of Z genes. We also show that LINE CR1 elements are enriched 2.7-fold on the chicken Z chromosome compared with autosomes and that chicken chromosomal length is highly correlated with percentage LINE content. However, the position of LINE elements is not significantly associated with dosage compensation status of Z genes. We also find a trend for a higher proportion of CpG islands in the region of the Z chromosome with the fewest dosage-compensated genes compared with the region containing the greatest concentration of compensated genes. Comparison between chicken and platypus genomes shows that LINE elements are not enriched on sex chromosomes in platypus, indicating that LINE accumulation is not a feature of all sex chromosomes. Our results suggest that CpG islands are not randomly distributed on the Z chromosome and may influence Z gene dosage compensation status.     

Identification and localization of two genes on the chicken Z chromosome: implication of evolutionary conservation of the Z chromosome among avian species

A cDNA clone containing an insert of about 3.4 kb, pCIREBP, was isolated from the chicken liver cDNA library and identified as a clone for the chicken homologue of iron-responsive element-binding protein (IREBP). The deduced amino acid sequence showed 88% identity with that of the mouse IREBP and 17 out of the 20 active site residues of the pig heart mitochondrial aconitase were conserved. Another cDNA clone, pZOV3, containing an insert of about 4.5 kb was isolated from the chicken ovary cDNA library. This cDNA contained an open reading frame for 327 amino acid residues, whose sequence had partial similarity to two immunoglobulin superfamily proteins; mouse GP-70 and chicken HT7. Fluorescencein situ hybridization using corresponding genomic clones revealed that both genes are localized on the Z chromosome; the ZOV3 gene at the middle of the short arm and the IREBP gene at the boundary of heterochromatin on the long arm. Southern blot hybridization to male and female genomic DNA preparations from six species representing five avian genera suggested that these two genes are Z-linked in all the species tested.     

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]₄, a microsatellite that also forms part of the pericentromeres, together with [GA]₈, [GATA]₄ and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.     

Various organizations of the complex repeats in vole sex chromosome heterochromatin

Different patterns of the DNA sequences organization were revealed in the vole (Rodentia) sex chromosome heterochromatin using dual-label fluorescence in-situ hybridization on extended DNA fibers with different repetitive DNA sequences as probes. In Microtus rossiaemeridionalis, the basic type represents the homogeneous relatively short tracks consisting of tandemly reiterated monomers of the MS3 family alternating with similar tracks of MS4 monomers and with non-fluorescent spacers. These tracks varied in the length of both repeats, with an average size of 12-22 kb or 3-5 copies. Apart from this, some continuous tracks of both families spanning 100-200 kb were interrupted by short spacers or single signals from the sequences with homology to LINEs. These results, together with that obtained by the analysis of phage clones of the genomic library, unequivocally demonstrate a variable large-scale DNA structural organization in heterochromatin of the M. rossiaemeridionalis sex chromosome. The dominant type of large-scale DNA organization in M. transcaspicus heterochromatin represents the unicolor relatively long tracks consisting of monotonous but not alternating monomers of MS3 or MS4 with sizes ranging from 15 to 40 kb and separated by extended spacers with an average length of 20 kb. Thus, the formation of the vole sex chromosome heterochromatic regions occurred relatively recently during speciation.     

Comparison of the chicken and zebra finch Z chromosomes shows evolutionary rearrangements

Using fluorescent in-situ hybridization (FISH) of zebra finch (Taeniopygia guttata) bacterial artificial chromosome (BAC) clones, we determined the chromosomal localizations of 14 zebra finch genes that are Z-linked in chickens: ATP5A1, CHD1, NR2F1, DMRT1, PAM, GHR, HSD17B4, NIPBL, ACO1, HINT1, SMAD2, SPIN, NTRK2 and UBE2R2. All 14 genes also map to the zebra finch Z chromosome, indicating substantial conservation of gene content on the Z chromosome in the two avian lineages. However, the physical order of these genes on the zebra finch Z chromosome differed from that of the chicken, in a pattern that would have required several inversions since the two lineages diverged. Eight of 14 zebra finch BAC DNA showed cross-hybridization to the W chromosome, usually to the entire W chromosome, suggesting that repetitive sequences are shared by the W and Z chromosomes. These repetitive sequences likely evolved in the finch lineage after it diverged from the Galliform lineage.     

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (<Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, Trionychidae)

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species     

A repeat DNA sequence from the Y chromosome in species of the genus <Emphasis Type="Italic">Microtus</Emphasis>

In most mammals, the Y chromosome is composed of a large amount of constitutive heterochromatin. In some Microtus species, this feature is also extended to the X chromosome, resulting in enlarged (giant) sex chromosomes. Several repeated DNA sequences have been described in the gonosomal heterochromatin of these species, indicating that it has heterogeneous and species-specific composition and distribution. We have cloned an AT-rich, 851-bp long, repeated DNA sequence specific for M. cabrerae Y chromosome heterochromatin. The analysis of other species of the genus Microtus indicated that this sequence is also located on the Y chromosome (male-specific) in three species (M. agrestis, M. oeconomus and M. nivalis), present on both Y and X chromosomes and on some autosomes in M. arvalis and absent in the genome of M. guentheri. Our data also suggest that the mechanism of heterochromatin amplification operating on the sex chromosomes could have been different in each species since the repeated sequences of the gonosomal heterochromatic blocks in M. cabrerae and M. agrestis are different. The absence of this sequence in the mouse genome indicates that its evolutionary origin could be recent. Future analysis of the species distribution, localization and sequence of this repeat DNA family in arvicolid rodent species could help to establish the unsolved phylogenetic relationships in this rodent group.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

Analysis of the cellular proto-oncogene mht/raf: relationship to the 5' sequences of v-mht in avian carcinoma virus MH2 and v-raf in murine sarcoma virus 3611

The avian carcinoma virus MH2 contains a hybrid gene delta gag-mht with a contiguous open reading frame of 2682 base pairs as well as v-myc and avian helper virus-related sequences. delta gag is a partial retroviral core protein gene while v-mht and v-myc are cell-drived sequences. The v-mht sequence can be divided into two regions: the v-raf-related region at its 3' end contains 969 nucleotides which are 94% related as amino acid sequence to the onc-specific v-raf sequence of murine sarcoma virus 3611 (MSV 3611), and the v-mht-specific region at its 5' end contains 173 nucleotides which are unrelated to either MSV 3611 or avian helper virus sequences. To study the origin of the v-mht-specific sequences, the 5' region of the proto-mht/raf gene was molecularly cloned from a phage lambda library containing genomic chicken sequences. Nucleic acid hybridization, heteroduplex and DNA sequence analyses indicate that the v-mht-specific sequences are encoded in three exons. The first and second exons are separated by a 3.4-kb intron while the second and third exons are separated by a 90-bp intron. The last 14 bp of the third exon are shared with v-raf and thus represent the start of v-raf-related sequences. The junction between v-mht-unrelated and related cellular sequences occurs within the first exon. There is no homology between the v-mht-unrelated sequences and the retroviral helper sequences indicating that the viral transduction of the proto-mht/raf sequences occurred through illegitimate recombination. The predominant v-mht-related messenger RNA (4.0 kb) hybridizes to several noncontiguous regions on the molecularly cloned cellular proto-mht/raf DNA indicating that the proto-mht/raf gene is distributed over at least 10 kb of DNA in the chicken genome. Thus the v-mht oncogene is a subset of its normal cellular homolog in that it lacks intervening sequences and possibly lacks 5'-coding sequences.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward