Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

Structural organization of alpha-satellite DNA in a single monkey chromosome

The organization of α-satellite sequences in a single monkey chromosome has been studied by restriction endonuclease analysis and molecular cloning. A somatic cell hybrid containing the monkey chromosome was isolated by cloning after fusion of the mouse L-cell line B82 (thymidine kinase minus) with primary African green monkey kidney cells and selective growth in HAT medium. Unlike the mouse cells, the hybrid cells contain DNA that hybridizes with the α-satellite DNA of the monkey. The presence of a single α-satellite containing monkey chromosome was demonstrated by Giemsa-11 staining and by the absence of both this chromosome and monkey α-satellite DNA sequences in cells after back-selection in bromodeoxyuridine. Hybridization of restriction endonuclease-digested hybrid cell DNA with a cloned segment of African green monkey α-satellite DNA showed distinctly different patterns from those observed with monkey total DNA. In particular, EcoRI and HaeIII restriction endonuclease sites are much more abundant in the satellite sequences in the thymidine kinase-carrying chromosome than they are in total satellite. A library of hybrid DNA was constructed in a λ bacteriophage. Analyses of purified recombinant phage that hybridized with α-satellite also indicated an abundance of EcoRI and HaeIII sites. Of nine phage studied in detail, no two showed identical distributions of the two restriction sites in the α-satellite sequences, suggesting the independent evolution of different domains within the single chromosome. These results indicate that the thymidine kinase-carrying chromosome contains distinct subsets (domains) of the α-satellite DNA of the whole monkey genome and further, that while the satellite sequence on the single chromosome is distinctive, it is also complex.     

The effect of natural selection on enzymic catalysis.

The karyotype of Drosophila nasutoides reveals a very large autosome pair at the metaphase plate. The application of the C-banding technique shows that this chromosome is almost entirely heterochromatic and an isochromosome (Cordeiro et al., 1975). Examination of the DNA isolated from purified nuclei of D. nasutoides in neutral CsCl gradients reveals four major satellites. As much as 60% of the total DNA appears as satellites in the DNA from larval brains. The buoyant densities of the four satellites, designated as I through IV in the order of descending density, are 1.687, 1.682, 1.669 and 1.665 g/cm³, respectively. All four satellites show strand separations in alkaline CsCl gradients with the least separation in satellite III. Thermal denaturation studies with purified native satellites show that satellites I and IV consist of repeats of identical sequences, whereas satellites II and III show a large sequence variation between repeating units. As much as 10 to 24% base-pair mis-matching is observed in the reassociated satellite II. The sequence complexities obtained from DNA reassociation kinetics data are 5, 103, 2.3 × 10⁶ and 46 nucleotide pairs for the satellites I, II, III and IV, respectively. The complexity of satellite III is almost as large as that of Escherichia coli, when the reassociation rate is corrected according to the amount of mis-matching in this satellite. All four satellite sequences are localized in one chromosome (dot chromosome) according to in situ hybridizations to polytene chromosomes. The large heterochromatic chromosome seen at the metaphase plate appears as the dot chromosome after polytenization. Therefore, the large heterochromatic chromosome contains all four satellite DNA components.     

Nap1 regulates proper CENP-B binding to nucleosomes

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25°?C and β-heterochromatic in X0 males at 14°?C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

Species-Specific Heterochromatin Prevents Mitotic Chromosome Segregation to Cause Hybrid Lethality in Drosophila

Postzygotic reproductive barriers such as sterility and lethality of hybrids are important for establishing and maintaining reproductive isolation between species. Identifying the causal loci and discerning how they interfere with the development of hybrids is essential for understanding how hybrid incompatibilities (HIs) evolve, but little is known about the mechanisms of how HI genes cause hybrid dysfunctions. A previously discovered Drosophila melanogaster locus called Zhr causes lethality in F1 daughters from crosses between Drosophila simulans females and D. melanogaster males. Zhr maps to a heterochromatic region of the D. melanogaster X that contains 359-bp satellite repeats, suggesting either that Zhr is a rare protein-coding gene embedded within heterochromatin, or is a locus consisting of the noncoding repetitive DNA that forms heterochromatin. The latter possibility raises the question of how heterochromatic DNA can induce lethality in hybrids. Here we show that hybrid females die because of widespread mitotic defects induced by lagging chromatin at the time during early embryogenesis when heterochromatin is first established. The lagging chromatin is confined solely to the paternally inherited D. melanogaster X chromatids, and consists predominantly of DNA from the 359-bp satellite block. We further found that a rearranged X chromosome carrying a deletion of the entire 359-bp satellite block segregated normally, while a translocation of the 359-bp satellite block to the Y chromosome resulted in defective Y segregation in males, strongly suggesting that the 359-bp satellite block specifically and directly inhibits chromatid separation. In hybrids produced from wild-type parents, the 359-bp satellite block was highly stretched and abnormally enriched with Topoisomerase II throughout mitosis. The 359-bp satellite block is not present in D. simulans, suggesting that lethality is caused by the absence or divergence of factors in the D. simulans maternal cytoplasm that are required for heterochromatin formation of this species-specific satellite block. These findings demonstrate how divergence of noncoding repetitive sequences between species can directly cause reproductive isolation by altering chromosome segregation.     

The location of DNA homologous to human satellite III DNA in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Radioactive RNA with sequences complementary to human DNA satellite III was hybridised in situ to metaphase chromosomes of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla) and the orangutan (Pongo pygmaeus). A quantitative analysis of the radioactivity, and hence of the chromosomal distribution of human DNA satellite III equivalent sequences in the great apes, was undertaken, and the results compared with interspecies chromosome homologies based upon Giemsa banding patterns. In some instances DNA with sequence homology to human satellite III is present on the equivalent (homologous) chromosomes in identical positions in two or more species although quantitative differences are observed. In other cases there appears to be no correspondence between satellite DNA location and chromosome homology determined by banding patterns. These results differ from those found for most transcribed DNA sequences where the same sequence is located on homologous chromosomes in each species.     

Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA

DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Simple Quantitative PCR Approach to Reveal Naturally Occurring and Mutation-Induced Repetitive Sequence Variation on the Drosophila Y Chromosome

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.     

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

Background and Aims

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification.

Methods

A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling.

Key Results

Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species.

Conclusions

The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.     

Origin and Differentiation of a Sex Chromosome System in Parodon hilarii (Pisces, Parodontidae). Satellite DNA, G- and C-banding

A satellite DNA sequence of Parodon hilarii (named pPh2004) was isolated, cloned and sequenced. This satellite DNA is composed of 200 bp, 60% AT rich. In situ hybridization (FISH) results revealed that the satellite DNA pPh2004 is located in the terminal regions of several chromosomes, forming highly evident blocks in some and punctual marks in others. The comparison between the FISH and C-banding results showed that the location of this satellite DNA coincides with that of most terminal heterochromatins. However, some regions are only marked by FISH whereas other regions are only marked by C-banding. The possible existence of more than one satellite DNA family could explain these partial differences. The in situ hybridization with the satellite DNA and the G- and C-bandings confirmed the presence of a sex chromosome system of the ZZ/ZW type in P. hilarii, as well as the correct identification of the Z chromosome in the karyotype. This chromosome displays a segment of terminal heterochromatin in the long arm, similar to the segment observed in the short arm of the W chromosome, also showing a G-banding pattern similar to that of the short arm and part of the long arm of the W chromosome. A hypothesis on the origin of the W chromosome from an ancestral chromosome similar to the Z chromosome is presented.     

The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes

We have investigated the large-scale organization of the human chAB4-related long-range multisequence family, a low copy-number repetitive DNA located in the pericentromeric heterochromatin of several human chromosomes. Analysis of genomic clones revealed large-scale (~100 kb or more) sequence conservation in the region flanking the prototype chAB4 element. We demonstrated that this low copy-number family is connected to another long-range repeat, the NF1-related (ΨNF1) multisequence. The two DNA types are joined by an ~2 kb-long tandem repeat of a 48-bp satellite. Although the chAB4- and NF1-like sequences were known to have essentially the same chromosomal localization, their close association is reported here for the first time. It indicates that they are not two independent long-range DNA families, but are parts of a single element spanning ~200 kb or more. This view is consistent both with their similar chromosomal localizations and the high levels of sequence conservation among copies found on different chromosomes. We suggest that the master copy of the linked chAB4–ΨNF1 DNA segment appeared first on the ancestor of human chromosome 17.     

Microdissection and Microcloning of Chromosome 5 in Gossypium arboreum

Construction of single chromosomal DNA libraries by chromosome micromanipulation is a useful tool for pursuing genomic studies. Thus far, micromanipulation in cotton has not been reported yet, which may be due to difficulty in preparing chromosomes of similar sizes. In this study, single chromosome micromanipulation was successfully achieved in cotton. A single chromosome 5 of Gossypium arboreum (cultivar Shixiya-1) carrying a large satellite at mitotic metaphase was isolated by microdissection using the Cell Cut Plus Laser micromanipulation system. The chromosomal DNA was digested by Sau 3A and ligated to Sau 3A linker adaptors. After two rounds of linker adaptor PCR (LA-PCR) amplification, DNA fragments ranging from 300 to 2,500?bp were acquired. Southern hybridization revealed that the PCR products had homology with genomic DNA of the cultivar Shixiya-1, indicating that DNA of chromosome 5 has been successfully amplified. The second round LA-PCR products and 45S rDNA and chromosome-specific BAC clones were used as probes for fluorescence in situ hybridization analysis on metaphase chromosome. The results confirmed that the LA-PCR products were derived from the isolated target chromosome. Hybridization signals of the second round LA-PCR products were mainly detected along the entire chromosome 5; in addition, weak signals were also observed on other chromosomes, indicating that there were some homologous nucleotide sequences in other chromosomes. The second round LA-PCR products were cloned to generate a chromosome-specific DNA library which contains approximately 173,000 clones. Evaluation based on 136 randomly selected clones showed that the size of the inserts varied from 500 to 1,800?bp with an average of 750?bp. The no-load rate was less than 1?%, the titer of the library was 1.2?×?10⁶ pfu mL^?1, and the rate of the single and low copy sequences was over 47?%. This library will facilitate specific probe screening, molecular mapping, gene cloning, and DNA sequencing for this chromosome.     

Alpha Satellite Insertion Close to an Ancestral Centromeric Region

Human centromeres are mainly composed of alpha satellite DNA hierarchically organized as higher-order repeats (HORs). Alpha satellite dynamics is shown by sequence homogenization in centromeric arrays and by its transfer to other centromeric locations, for example, during the maturation of new centromeres. We identified during prenatal aneuploidy diagnosis by fluorescent in situ hybridization a de novo insertion of alpha satellite DNA from the centromere of chromosome 18 (D18Z1) into cytoband 15q26. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by the lack of constriction and the absence of CENP-A binding. The insertion was associated with a 2.8-kbp deletion and likely occurred in the paternal germline. The site was enriched in long terminal repeats and located ∼10 Mbp from the location where a centromere was ancestrally seeded and became inactive in the common ancestor of humans and apes 20–25 million years ago. Long-read mapping to the T2T-CHM13 human genome assembly revealed that the insertion derives from a specific region of chromosome 18 centromeric 12-mer HOR array in which the monomer size follows a regular pattern. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the methylation status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name “alpha satellite insertion.” It also expands our knowledge on alphoid DNA dynamics and conveys the possibility that alphoid arrays can relocate near vestigial centromeric sites.     

Banding in Human Chromosomes treated with Trypsin

THE differential staining properties of the Giemsa stain were first observed by Pardue and Gall¹. They were studying in situ hybridization between mouse satellite DNA and mouse chromosomes and observed that following certain pretreatment the centromeric regions of mouse chromosomes were more densely stained by Giemsa stain than other regions. The darkly stained regions were considered to consist of constitutive heterochromatin. Similar observations were later made on human chromosomes by Arrighi and Hsu² and Gagné et al.³. Through modifications of the original methods used in the DNA hybridization work, techniques have been developed which make each chromosome identifiable^4–6.     

Locations of a Human Satellite DNA in Human Chromosomes

USING techniques for DNA/RNA or DNA/DNA hybridization in situ, Pardue and Gall¹ and Jones² made several significant discoveries on the chromosomal locations of the mouse satellite DNA: (1) this fraction of DNA is found in all chromosomes except the Y, (2) the cytological location of the satellite DNA is limited to the centromeric region of each chromosome and is probably absent in other regions and (3) the centromeric regions of all mouse chromosomes are hetero-chromatic.     

Chromosome banding and DNA replication studies on a cell line of Dipodomys merriami

The chromsomes of a cell line of Dipodomys merriami are described in terms of their C-, G- and Q-banding patterns. Studies on the buoyant density of DNA made at different times in the S phase show that the replication of HSα satellite and AT-rich main band DNA occurs preferentially late in the S phase, whereas MS satellite and GC-rich main band DNAs are replicated early in the S phase. Autoradiographic studies of chromosomes labelled early or late in the S phase are used to relate the banding patterns nf particular chromosome regions to the fraction of DNA which they may contain.