Human genome dispersal and evolution of 4q35 duplications and interspersed LSau repeats

We have investigated the evolutionary history of the 4q35 paralogous region, and of a sub-family of interspersed LSau repeats. In HSA, 4q35 duplications were localized at 1q12, 3p12.3, 4q35, 10q26, 20cen, whereas duplicons and interspersed LSau repeats simultaneously labeled the p arm of acrocentric chromosomes. A multi-site localization of 4q35-like sequences was also observed in PTR, GGO, PPY, HLA (Hominoidea) and PAN (Old World monkey), thus indicating that duplications of this region have occurred extensively in the two clades, which diverged at least 25 million years ago. In HSA, PTR and PAN, 4q35-derived duplicons co-localized with rDNA, whereas in GGO and PPY this association was partially lacking. In PAN, the single- and multi-site distribution of rDNA and paralogous sequences, respectively, indicates a different timing of sequence dispersal. The sub-family of interspersed LSau repeats showed a lesser dispersal than 4q35 duplications both in man and great apes. This finding suggests that duplications and repeated sequences have undergone different expansion/contraction events during evolution. The mechanisms underlying the dispersal of paralogous regions may be further derived through studies comparing the detailed structural organization of these genomic regions in man and primates.     

Trisomy 8p: unusual origin detected by fluorescence in situ hybridization

Summary Chromosomal analysis of a neonate with brain and heart abnormalities revealed trisomy for 8p. The mother's karyotype showed 47 chromosomes with one chromosome 8 being represented as two separate chromosomes, an acrocentric 8p and a telocentric 8q. Gbanding and silver staining revealed a satellite and nucleolus organizing region (NOR) on the 8p. Centromericspecific probes to the centromeres of chromosomes 8, 15, 13/21, 22 and the acrocentric chromosomes revealed that only the 8q centromere was of chromosome-8 origin, while the 8p centromere was of chromosome-14 origin.     

A homologous subfamily of satellite III DNA on human chromosomes 14 and 22.

We describe a new subfamily of human satellite III DNA that is represented on two different acrocentric chromosomes. This DNA is composed of a tandemly repeated array of diverged 5-base-pair monomer units of the sequence GGAAT or GGAGT. These monomers are organised into a 1.37-kilobase higher-order structure that is itself tandemly reiterated. Using a panel of somatic cell hybrids containing specific human chromosomes, this higher-order structure is demonstrated on chromosomes 14 and 22, but not on the remaining acrocentric chromosomes. In situ hybridisation studies have localised the sequence to the proximal p-arm region of these chromosomes. Analysis by pulsed-field gel electrophoresis (PFGE) reveals that 70-110 copies of the higher-order structure are tandemly organised on a chromosome into a major domain which appears to be flanked on both sides by non-tandemly repeated genomic DNA. In addition, some of the satellite III sequences are interspersed over a number of other PFGE fragments. This study provides fundamental knowledge on the structure and evolution of the acrocentric chromosomes, and should extend our understanding of the complex process of interchromosomal interaction which may be responsible for Robertsonian translocation and meiotic nondisjunction involving these chromosomes.     

Breakpoints in Robertsonian translocations are localized to satellite III DNA by fluorescence in situ hybridization.

We characterized 21 t(13;14) and 3 t(14;21) Robertsonian translocations for the presence of DNA derived from the short arms of the translocated acrocentric chromosomes and identified their centromeres. Nineteen of these 24 translocation carriers were unrelated. Using centromeric alpha-repeat DNA as chromosome-specific probe, we found by in situ hybridization that all 24 translocation chromosomes were dicentric. The chromatin between the two centomeres did not stain with silver, and no hybridization signal was detected with probes for rDNA or beta-satellite DNA that flank the distal and proximal ends of the rDNA region on the short arm of the acrocentrics. By contrast, all 24 translocation chromosomes gave a distinct hybridization signal when satellite III DNA was used as probe. This result strongly suggests that the chromosomal rearrangements leading to Robertsonian translocations occur preferentially in satellite III DNA. We hypothesize that guanine-rich satellite III repeats may promote chromosomal recombination by formation of tetraplex structures. The findings localize satellite III DNA to the short arm of the acrocentric chromosomes distal to centromeric alpha-repeat DNA and proximal to beta-satellite DNA.     

赤斑羚和斑羚核型比较研究

本文对赤斑羚（ＮａｅｍｏｒｈｅｄｕｓＣｒａｎｂｒｏｏｋｉ）和斑羚（Ｎ．ｇｏｒａｌｇｒｉｓｅｕｓ）的染色体Ｇ带、Ｃ带和Ａｇ－ＮＯＲｓ的数目、分布等作了较详细的比较研究。赤斑羚２ｎ＝５６全部为近端着丝粒染色体,Ｎ．Ｆ＝５４;斑羚２ｎ＝５４,除Ｎｏ．３是亚中着丝粒染色体外,具有丰富的异染色质;二者Ｇ带带纹相似程度高,其Ｎｏ．３长臂Ｇ带带纹相似。斑羚的Ｎｏ．３短臂与赤斑羚Ｎｏ．２７近端着丝粒染色体的大小、     

RHG-band polymorphism of the short arms of human acrocentric chromosomes and relationship of variants to satellite associations

Summary The extent of RHG-band variation of short arms of human acrocentric chromosomes was investigated in a group of 100 subjects by visually comparing the variants with the size of reference bands 7p22, 21q22 and 11q13. Marked differences were found among the chromosomes in the distribution of variants; the largest mean size of RHG-band was associated with chromosome 21, whereas the variants of chromosome 22 had the smallest band size. The study further showed that the involvement of acrocentric chromosomes in satellite association did not depend upon the size of RHG-band variants.     

Chromosome 15 anomalies and the Prader-Willi syndrome: Cytogenetic analysis

Summary The behaviour of chromosome 15 is very different from that of the other acrocentric chromosomes. The cytogenetic characteristics of rearrangements associated with Prader-Willi syndrome (PWS) are analyzed as similar rearrangements irrespective of the associated phenotype (reciprocal translocations of chromosome 15, small bisatellited additional chromosomes, Robertsonian translocations, interstitial deletions, pericentric inversions). This study suggests that: (1) The proximal (15q) region and PWS seem to be indissociable; (2) chromosome 15 has an indisputable cytogenetic originality which could be related to its histochemical properties. Chromosome 15 constitutive heterochromatin usually contains much 5-methylcytosine-rich DNA and a large amount of each of the four satellite DNAs. Furthermore the existence in the proximal (15q) region of one or several palindromic sequences could be postulated to explain the great lability of this region of chromosome 15.     

Quantitative studies on the arrangment of human metaphase chromosomes

Summary The pattern of association of acrocentric chromosomes was examined in ten and five carriers of a 15/21 and a 13/14 Robertsonian translocation, respectively, and was compared with that of the same numbers of relatives with normal karyotypes. In the carriers of 15/21 translocation, the number of large associations (involving more than two acrocentrics) and the association frequencies for individual acrocentric chromosomes, were significantly higher than in the control group. The mean number of associations of the single homologs of the translocation chromosomes was much higher than that of the other acrocentrics. In the carriers of 13/14 translocations, only the association frequency for chromosome 13 was higher than in the normal relatives. The uninvolved chromosomes homologous to those involved in translocations showed an insignificant increase in associations in comparison with the other acrocentrics. These results suggest that some mechanism within the cells compensates for the effect of missing acrocentrics or of acrocentrics lacking NORs on the number of associations. The possible relations of this phenomenon to the activity of the nucleolus organizing regions are discussed.     

Using Alu J Elements as Molecular Clocks to Trace the Evolutionary Relationships Between Duplicated HLA Class I Genomic Segments

The class I region of the major histocompatibility complex contains two subgenomic blocks (250–350 kb each), known as the alpha and beta blocks. These blocks contain members of multicopy gene families including HLA class I, HERV-16 (previously called P5 sequences), and PERB11 (MIC). We have previously shown that each block consists of imperfect duplicated segments (duplicons) containing linked members of different gene families, retroelements and transposons that have coevolved as part of two separate evolutionary events. Another region provisionally designated here as the kappa block is located between the alpha and the beta blocks and contains HLA-E, -30, and -92, HERV-16 (P5.3), and PERB11.3 (MICC) within about 250 kb of sequence. Using Alu elements to trace the evolutionary relationships between different class I duplicons, we have found that (a) the kappa block contains paralogous (duplicated) Alu J sequences and other retroelement patterns more in common with the beta than the alpha block; (b) the retroelement pattern associated with the HLA-E duplicon is different from all other HLA class I duplicons, indicating a more complex evolution; (c) the HLA-92 duplicon, although substantially shorter, is closely related in sequence to the HLA-B and -C duplicons; (d) two of the six paralogous Alu J elements within the HLA-B and -C duplicons are associated with the HLA-X duplicon, confirming their evolutionary relationships within the beta block; and (e) the paralogous Alu J elements within the alpha block are distinctly different from those identified within the beta and kappa blocks. The sequence conservation and location of duplicated (paralogous) Alu J elements in the MHC class I region show that the beta and kappa blocks have evolved separately from the alpha block beginning at a time before or during the evolution of Alu J elements in primates. Received: 22 September 1999 / Accepted: 24 January 2000     

Sequential staining for G- and C-banding of chromosomes in the analysis of the morphology of the short arms of human acrocentric chromosomes]

Sequential staining for G- and C-banding of acrocentric chromosomes of 8 persons showed that the large heterochromatin region occurred more frequently in chromosome 15 than in chromosomes 13 and 14, and in chromosome 22 more frequently than in chromosome 21. There proved to be no correlation between the size of the heterochromatic region and the short arm of the acrocentric chromosomes. The frequency of occurrence of the satellites in the 8 persons was approximately the same for all the acricentric pairs. The C-banded satellite region of the homologous chromosomes is often heteromorphic.     

Patterns of association in the human metaphase complement: Ring analysis and estimation of associativity of specific chromosome regions

Summary The pattern of metaphase chromosome association in the human complement was studied by two methods of statistical analysis of interchromosomal distances. Those methods included ring analysis in which a characteristic position of the centromere of each chromosome relative to the center of a two-dimensional representation of a metaphase complement was defined, and estimation of the capacity for associativity of each of three regions of each chromosome: the centromere (c) and the ends of each arm (p,q).The following information was obtained: 1. In general, the distance from the center is directly related to chromosome size. 2. The most notable deviation from that size-related progression is displayed by the X chromosomes. The markedly peripheral position of the X is characteristic of both X's of the female and the single X of the male. 3. The relative associativity of each chromosome of the complement is, in general, inversely related to size with an additional preferential capacity of associativity displayed by the acrocentric chromosomes. Analyses of the different inter-regional classes established that the supplementary associativity factor of the acrocentric chromosomes was inherent in their pericentromeric and p-arm regions and excluded the ends of the q arms from participation in that factor. 4. Those analyses demonstrated that the specific morphology or geometry of the acrocentric chromosomes contributes little to their high relative associativity. In addition to the tendency for the c/p regions of the acrocentric chromosomes to associate with each other, presumably because of their common function in nucleolar organization, those regions also displayed a propensity to associate with the distal regions of the arms of other chromosomes. A molecular basis for that propensity other than that of ribosomal DNA is postulated to be that of other fractions of highly reiterated DNA sequences. 5. Analysis of the relative associativities of each of the three regions of the Y chromosome revealed that the Yq displays a much stronger capacity to associate with the c's of other chromosomes than does the Yc or Yp.     

Molecular cytogenetic characterization of 17 rob(13q14q) Robertsonian translocations by FISH, narrowing the region containing the breakpoints.

We have characterized 17 rob(13q14q) Robertsonian translocations, using six molecular probes that hybridize to the repetitive sequences of the centromeric and shortarm regions of the five acrocentric chromosomes by FISH. The rearrangements include six de novo rearrangements and the chromosomally normal parents, five maternally and three paternally inherited translocations, and three translocations of unknown origin. The D21Z1/D13Z1 and D14Z1/D22Z1 centromeric alpha-satellite DNA probes showed all rob(13q14q) chromosomes to be dicentric. The rDNA probes did not show hybridization on any of the 17 cases studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 was retained around the breakpoints in all cases. However, the pTRS-63 satellite III DNA probe specific for chromosome 14 did not show any signals on the translocation chromosomes examined. In 16 of 17 translocations studied, strong hybridization signals on the translocations were detected with the pTRI-6 satellite I DNA probe specific for chromosome 13. All parents of the six de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong positive hybridization signals on each pair of chromosomes 14 and 13, with pTRS-47, pTRS-63, and pTRI-6. Therefore, the translocation breakpoints in the majority of rob(13q14q) are between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13.     

Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.

We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.     

Immunocytochemical analysis of human metaphase chromosome methylation status

The present paper describes a distribution of 5-methylcytosine-rich DNA in human metaphase chromosomes from PHA-stimulated lymphocytes. Immunocytochemical detection of 5-methylcytosine was carried out with monoclonal antibodies. Fluorescent signals were preferentially localized in certain chromosomal regions, corresponding to R-, some T-bands, pricentromeric heterochromatin, and short arms of acrocentric chromosomes. Specificity of fluorescent signals distribution along chromosomes allowed to describe a new type of human metaphase chromosomes banding pattern, which we call M-banding. Specific M-markers of landmarks were identified for each chromosome pair. The analysis of M-bands methylation status was carried out taking into account data available in literature on their nucleotide structure features, namely GC-rich H3 isochore content and CpG-islands concentration. According to our results, a high level of methylation is typical for the majority of GC-rich regions. However, certain bands of 6, 9, 10, 13, 15 chromosomes (6q15, 6q21, 6q23, 9p13, 9p22, 9p32, 10q24, 13q22, 15q15, 15q24) were shown to be hypomethylated, suggesting their special functional status in lymphocytes.     

Recent evolution of DNA sequence homology in the pericentromeric regions of human acrocentric chromosomes

A search for genes located on human chromosome 21 resulted in the isolation of a HeLa cDNA clone, pUNC724, which hybridized to 3.7 and 2.5 kilobase (kb) EcoRI fragments on each of the human acrocentric chromosomes. In situ hybridization further localized pUNC724 to the pericentromeric region of the human acrocentrics. Two other EcoRI fragments that hybridized to pUNC724 were assigned to the long arms of chromosomes 1 and 18. The pUNC724 sequence does not appear to be related to ribosomal or satellite DNA sequences. The juxtaposition of DNA sequences homologous to pUNC724 and ribosomal DNA sequences presumably occurred within the past thirty-five million years, following the divergence of the lines leading to man and the New World owl monkey, Aotus trivirgatus--pUNC724 is not syntenic with the single chromosome containing ribosomal DNA sequences in the owl monkey.     

Relationship between measured chromosome distribution parameters and Ag-staining of the nucleolus organizer regions

Summary Trypsin-banded metaphase plates provided by one whole blood culture of a normal adult female were analyzed as to the chromosome distribution by measuring: (1) distances between centromeres; (2) angles formed between a centromere, the gravity center of the metaphase plane, and a second centromere; and (3) the measured tendency to associate, as defined by Galperin (1969b). These data are correlated with Ag-NOR staining findings obtained from 72 cells from another culture of the same individual. In these cells, the chromosome pairs are identified using a simultaneous Ag-NOR staining and acridine orange banding technique. The silver precipitation is also correlated with the scored satellite associations in these cells. The results show a correlation between all concerned parameters, indicating that the nucleolar function of the human acrocentric chromosomes, as demonstrated by the silver precipitation technique, is probably one of the major determinants of the proximity of these chromosomes. There is a pronounced correlation of the Ag-NOR findings with those measured parameters which describe best the preferential small distances between chromosomes (angle analysis and tendency to associated data). Moreover, the association patterns of the acrocentrics with small amounts of NOR provide some evidence for the interference of other determinants cogoverning the position of the human D-and G-group chromosomes.To whom offprint requests should be sent     

A set of duplicons on human chromosome 9 is involved in the origin of a supernumerary marker chromosome

Human chromosome 9 is involved in a number of recurrent structural rearrangements; moreover, its pericentromeric region exhibits a remarkable evolutionary plasticity. In this study we present the molecular characterization of a constitutional rearrangement, involving the 9p21.1q13 region, which led to the formation of a supernumerary marker chromosome (SMC). We defined the sequence of the breakpoints and identified a new set of duplicons on human chromosome 9, named LCR9s (chromosome 9 low-copy repeats). Two of these duplicons were shown to be involved in a somatic exchange leading to the formation of the SMC. High-resolution FISH coupled to database search demonstrated that a total number of 35 LCR9 paralogs are present in the human genome. These newly described chromosome 9 duplicons have features that may be crucial in driving structural chromosome rearrangements in germinal and somatic cells.     

Heterochromatic Effects on the Behavior of Reversed Acrocentric Compound-X Chromosomes in DROSOPHILA MELANOGASTER

Previous studies of reversed acrocentric compound-X chromosomes suggested peculiar influences of heterochromatin on both the synthesis and meiotic behavior of such compunds. It seemed, with respect to synthesis, that the long arm of the Y chromosome on an X.Y(L) chromosome was necessary in order for the heterochromatic exchange giving rise to reversed acrocentrics to occur, even though Y(L) itself did not participate in the compound-generating event. With respect to behavior, the resulting compounds appeared, presumably as a consequence of their singular generation, to contain an interstitial heterochromatic region that caused the distribution of exchanges between the elements of the compound to be abnormal (many zero and two-exchange tetrads with few, if any, single-exchange tetrads). Removing the intersititial heterochromatin (or, curiously, appending Y(L) as a second arm of the compound) eliminated the recombinational anomalies and resulted in typical tetrad distributions.--We provide evidence that these peculiarities, while presumably real, were likely the consequence of a special X.Y(L) chromosome that was used to synthesize the reversed acrocentrics examined in the early studies and are not general properties of either reversed acrocentric compounds or of interstitial heterochromatin. However, we show that specific heterochromatic regions do, in fact, profoundly influence the behavior of (apparently all) reversed acrocentric compound-X chromosomes. In particular, we demonstrate that specific portions of the Y chromosome and of the basal X-chromosome heterochromatin, when present as homologs for reversed acrocentric compounds, markedly and coordinately increase both the frequency of exchange between the elements of the compound and the fertility (egg production) of compound-bearing females. It is, we suppose, some aspect of this heterochromatic effect, produced by the special X.Y(L) chromosome, that caused the earlier-analyzed compounds to exhibit the observed anomalies.     

Chromosome banding homologies in Swamp and Murrah buffalo

Silver staining of Swamp buffalo (2n = 48) metaphase chromosomes revealed telomeric nucleolus organizer regions (NOR's) located on five pairs of autosomes identified by R-banding as numbers 4 p (submetacentric), 8, 20, 22, and 23 (acrocentrics); interphase nuclei also showed no more than five nucleoli. The Murrah buffalo (2n = 50) was previously reported to have telomeric NOR's located on six pairs, -3 p and 4 p (submetacentric), 8, 21, 23, and 24 (acrocentrics). By comparing the two types of buffalo it was concluded that: all of the chromosomes are similar in banding patterns; chromosome 1 of Swamp results from a telomere-centromere tandem fusion between two chromosomes identified as 4 p and 9, respectively, in the Murrah karyotype, thus accounting for the reduced diploid number of Swamp buffalo; the fusion causes the loss of NOR's on the telomeres of chromosome 4, thus accounting for the reduced number of NOR chromosome pairs of Swamp; the presence of a pale C-band are in the region of junction between chromosome 4 and 9 involved in the fusion suggests that the centromeric region of the later is retained and altered.