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S H Cross

Publications and source records attributed to S H Cross.

14 recordsLinked to original sources
PubMed2000

CpG island libraries from human chromosomes 18 and 22: landmarks for novel genes.

CpG islands are found at the 5' end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5' end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5' end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5' gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5' gene sequences from specific human chromosomes.

Base Sequence↗
PubMed1999

Isolation of CpG islands from large genomic clones.

Positional cloning is a powerful method for the identification of genes. Using genetic and physical mapping methods the genomic region within which a particular gene is located can relatively easily be narrowed down to a comparatively small area contained within cosmid, PAC or BAC clones. It is then a matter of identifying genes within these clones. Here we describe the appli-cation of a technique, which has been successfully used for the bulk purification of CpG islands from whole genomes, to the isolation of CpG island sequences from such clones. As CpG islands overlap transcription units they can be used to isolate full-length cDNAs for associated genes, either by probing cDNA libraries or by searching databases. CpG islands are linked with approximately 60% of human genes and because their isolation is independent of the expression profile of these genes this approach would complement other expression-based methods of gene identification. By applying this technique to a cosmid clone known to contain the PAX6 gene we successfully isolated the CpG island for this gene along with other CpG island-like sequences. Closer examination revealed that an extensive genomic region around the 5'-end of PAX6 is unusual with regard to methylation and GC content. CpG island sequences were also successfully isolated from a PAC clone carrying the MBD1 gene. These included the complete CpG island containing the first exon and regulatory sequences from MBD1.

Base Sequence↗
PubMed1997

The chromosomal distribution of CpG islands in the mouse: evidence for genome scrambling in the rodent lineage.

We have prepared a library of mouse whole CpG islands using a methyl-CpG binding domain column. The distribution of CpG islands in the mouse genome was determined by FISH, using the library as a probe. Unlike in other vertebrate genomes that have been examined (human and chicken), extreme clustering of CpG islands was not seen in the mouse genome. No individual murine chromosome stood out as being either very gene-rich or very gene-poor. Despite the more even distribution of CpG islands in the mouse at a gross chromosomal level, at finer resolution concentrations of CpG islands are seen to correspond to the R-band early replicating regions of the genome.

Animals↗
PubMed1997

A component of the transcriptional repressor MeCP1 shares a motif with DNA methyltransferase and HRX proteins.

Methylation of cytosines within the sequence CpG is essential for mouse development and has been linked to transcriptional suppression in vertebrate systems. Methyl-CpG binding proteins (MeCPs) 1 and 2 bind preferentially to methylated DNA and can inhibit transcription. The gene for MeCP2 has been cloned and a methyl-CpG binding domain (MBD) within it has been defined. A search of DNA sequence databases with the MBD sequence identified a human cDNA with potential to encode an MBD-like region. Sequencing of the complete cDNA revealed that the open reading frame also encodes two cysteine-rich domains that are found in animal DNA methyltransferases (DNMTs) and in the mammalian HRX protein (also known as MLL and All-1). HRX is related to Drosophila trithorax. The protein, known as Protein Containing MBD (PCM1), was expressed in bacteria and shown to bind specifically to methylated DNA. PCM1 also repressed transcription in vitro in a methylation-dependent manner. A polyclonal antibody raised against the protein was able to 'supershift' the native MeCP11 complex from HeLa cells, indicating that PCM1 is a component of mammalian MeCP1.

Amino Acid Sequence↗
PubMed1996

CpG islands of chicken are concentrated on microchromosomes.

The chicken karyotype comprises 39 chromosome pairs of which at least 29 are 'microchromosomes'. Microchromosomes account for about 25% of the genomic DNA, but they are cytologically indistinguishable from one another (1). Due to technical limitations there is a strong bias of mapped genes within the chicken genome database ChickGBASE (2) towards macrochromosomes 1-6 and Z, with specific assignments to only one microchromosome (3,4). Several genes have, however, been assigned to the microchromosome group as a whole (3,5-9), demonstrating that these tiny chromosomes do not represent genetically inert DNA. To determine the overall chromosomal distribution of genes, as well as to provide a mapping resource, we prepared a CpG island library from chicken using differential binding to a methyl-CpG chicken using differential binding to a methyl-CpG binding column before and after de novo methylation (10). Surprisingly, we found that chicken CpG islands are highly concentrated on the microchromosomes, whereas macrochromosomes 1-6 are comparatively gene-poor by this assay. Our results raise the possibility that gene density on chicken microchromosomes approaches the maximum value known for vertebrates.

Animals↗
PubMed1995

CpG islands and genes.

Of the estimated 45,000 CpG islands in the human genome, the overwhelming majority are found at the 5' ends of genes and their identification and cloning are proving very useful for finding and isolating genes. Recent work has shed light on the chromosomal distribution and origin of CpG islands. It has been shown unequivocally that CpG islands are concentrated in the R band chromosomal regions and that intact transcription factor binding sites and required for their maintenance. Cases of methylation of CpG islands and inactivation of the associated genes have been reported which may be important in ageing, tumorigenesis and imprinting.

Animals↗
PubMed1994

Genetic and physical mapping of a gene encoding a methyl CpG binding protein, Mecp2, to the mouse X chromosome.

The methyl CpG binding proteins (MeCP1 and MeCP2) are a class of proteins that bind to templates containing symmetrically methylated CpGs. Using an interspecific backcross segregating a number of X-linked markers, we have localized the Mecp2 gene in mouse to the X chromosome close to the microsatellite marker DXMit1. Detailed physical mapping utilizing an available YAC contig encompassing the DXMit1 locus has localized the Mecp2 gene to a 40-kb region between the L1cam and the Rsvp loci, indicating the probable position of a homologue on the human X chromosome.

Animals↗
PubMed1994

Purification of CpG islands using a methylated DNA binding column.

CpG islands are short stretches of DNA containing a high density of non-methylated CpG dinucleotides, predominantly associated with coding regions. We have constructed an affinity matrix that contains the methyl-CpG binding domain from the rat chromosomal protein MeCP2, attached to a solid support. A column containing the matrix fractionates DNA according to its degree of CpG methylation, strongly retaining those sequences that are highly methylated. Using this column, we have developed a procedure for bulk isolation of CpG islands from human genomic DNA. As CpG islands overlap with approximately 60% of human genes, the resulting CpG island library can be used to isolate full-length cDNAs and to place genes on genomic maps.

Amino Acid Sequence↗
PubMed1989

Cloning of human telomeres by complementation in yeast.

Telomeres confer stability on chromosomes by protecting them from degradation and recombination and by allowing complete replication of the end. They are genetically important as they define the ends of the linkage map. Telomeres of lower eukaryotes contain short repeats consisting of a G-rich and a C-rich strand, the G-rich strand running 5'-3' towards the telomere and extending at the end. Telomeres of human chromosomes share characteristics with those of lower eukaryotes including sequence similarity as detected by cross-hybridization. Telomeric repeats from many organisms can provide telomere function in yeast. Here we describe a modified yeast artificial chromosome (YAC) vector with only one telomere which we used to clone human telomeres by complementation in yeast. YACs containing human telomeres were identified by hydridization to an oligonucleotide of the trypanosome telomeric repeat. A subcloned human fragment from one such YAC is immediately subtelomeric on at least one human chromosome.

Animals↗
PubMed1988

Telomeric repeat from T. thermophila cross hybridizes with human telomeres.

The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.

Animals↗
PubMed1986

A cosmid vector for systematic chromosome walking.

We describe the construction of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA polymerase promoter sequences that are oriented towards and immediately adjacent to HindIII and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment of steps in a cosmid walk.

Base Sequence↗
PubMed1985

A cosmid vector that facilitates restriction enzyme mapping.

We describe the construction and use of a cosmid vector, loric, which is derived from the phage lambda origin of replication and appears to be more stable than ColE1-derived cosmids. Loric recombinants can be efficiently packaged in vivo to yield 100-300 micrograms of DNA per liter that is linear and has single-stranded cos ends. We call such molecules "phosmids." Phosmid restriction maps can be rapidly generated by labeling either the left or right cos site by annealing on a 32P-labeled oligonucleotide complementary to either cos-L or cos-R. Partial restriction enzyme digestion, agarose gel electrophoresis, and autoradiography are used to size restriction fragments of increasing length, all of which terminate at the labeled cos site. The procedures have been tested by isolating and mapping a region of the H-2 locus of mouse chromosome 17.

Bacteriophage lambda↗