Arthur R. Mitchell

A cloned sequence, p82H, of the alphoid repeated DNA family found at the centromeres of all human chromosomes.

Clone p82H is a human DNA sequence which hybridises in situ exclusively to the centromeric regions of all human chromosomes. It is composed of approximately 14 tandemly repeated variants of a basic 172 bp sequence, and is related to the alphoid family. The organisation of the family of cross-hybridising sequences, detected by the clone p82H, is described both in the human genome and on certain chromosomes, and its relationship to known sequence families is discussed.

Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes

Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by “stretching” the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B.

Specific arrangements of human satellite III DNA sequences in human chromosomes.

DNA was extracted from various rodent-human somatic cell hybrids that contained single or a few human chromosomes. These DNAs were examined by a combination of restriction endonuclease digestion, gel electrophoresis, and filter hybridisation to radioactive satellite DNA probes following transfer of the denatured restriction fragments from a gel to a nitrocellulose filter. In this way the arrangement of sequences homologous to human satellite III were examined on human chromosomes 1, 7, 11, 15, 22 and X. It was found that the distribution of restriction endonuclease sites within satellite III DNA is different on different chromosomes.

Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite.

Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consisten with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.

The organisation of repetitive DNA sequences on human chromosomes with respect to the kinetochore analysed using a combination of oligonucleotide primers and CREST anticentromere serum

The spatial relationship between the families of repetitive DNAs present at the centromeres of human chromosomes and the position of the kinetochore was examined by combining immunocytochemistry with the PRINS oligonucleotide primer extension technique. Heterochromatic domains were decondensed with 5′-azacytidine to facilitate this study. Using this approach our results clearly show that the alphoid DNA sequences are closely associated with the kinetochore of human chromosomes. Simple-sequence satellite DNAs occupy separate, non-overlapping domains within the centromere. These two major families are separated by a third, relatively low-copy repetitive DNA family, SAU-3A. Pulse-field gel electrophoresis was employed to analyse the centromeric domain of human chromosome no. 9 in more detail and the results although preliminary support the conclusions drawn from the immunocytochemistry/PRINS approach.

Specificity of SAF‐A and lamin B binding in vitro correlates with the satellite DNA bending state

There is evidence that Matrix Attachment Region (MAR)‐binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR‐binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin κ gene (Ig κ MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA‐binding proteins were identified as SAF‐A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF‐A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [ 1987 ] Nature 326:720–722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF‐A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR‐binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments. J. Cell. Biochem. 83: 218–229, 2001.

A chimpanzee-derived chromosome-specific alpha satellite DNA sequence conserved between chimpanzee and human.

We describe a cloned 2.7 kb alpha satellite sequence, Pan-3, from the pygmy chimpanzee (Pan paniscus) that specifically hybridizes in situ to chromosome 19 in the pygmy chimpanzee and to the homeologous human chromosome, no. 17. Using high stringency conditions of hybridization on Southern blots, this sequence hybridized to DNA from both species of chimpanzee (P. paniscus and P. troglodytes) and from human but not to DNA from gorilla (Gorilla gorilla) or orangutan (Pongo pygmaeus). Partial sequence analysis showed that Pan-3 and a previously described human chromosome 17-specific clone have up to 91% sequence identity. To our knowledge this is the highest sequence similarity reported between alphoid subsets from human and any other primate.

Nuclear envelope associated protein that binds telomeric DNAs.

Rana temporaria oocytes at the 6th diplotene stage of maturation contain a special structure, the karyosphere capsule, with chromosomes covered and detached from the nuclear envelope (NE), though at the previous stage the telomeres were attached to the membrane, as characteristic of germ cells. The DNA–protein complexes from band shift assays with proteins extracted from oocyte NEs and telomeric DNA fragment (T2G4)130 were isolated and injected into a guinea pig. In the present paper the only protein of 70 kDa recognized by antibody (AB) in the NE is named the Membrane Telomere Binding Protein (MTBP). Western blots with guinea pig AB and AB against telobox peptide from TRF2 show that protein of 60 kDa (probably TRF1) belongs to the chromatin, but MTBP (TRF2 according to immunoprecipitation) belongs to the NE. In the somatic cell nuclei both proteins are present and recognized by AB against telobox peptide, but AB raised recognize only MTBP/TRF2 due to the epitope different from telobox. Combined in situ hybridization with the vertebrate telomeric DNA sequences (T2AG3)135 and immunocytochemistry with the MTBP AB showed them to be colocalized within the mouse nucleus. As it was shown by immunofluorescense of NE spread, MTBP is organized in a distinct pattern that looks like a network made of double‐dots. Electron microscope immunogold staining with both ABs showed that the protein is localized on the outer surface of the oocyte NE within cup‐like structures attached to the membrane. This is the first clear evidence of a protein, which could be responsible for the attachment of telomeres to the nuclear membrane. Mol. Reprod. Dev. 57:16–25, 2000.

Restriction endonuclease/nick translation of fixed mouse chromosomes: A study of factors affecting digestion of chromosomal DNA in situ

We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.