Biomedical subjects
Publications and source records attributed to J F Cheng.
Brianolide (1), a new antiinflammatory diterpenoid of the briarein class, possessing a beta substituent at C-12 (R), has been isolated from the Okinawan gorgonian Briareum sp. Its structure has been established from spectral data in conjunction with a single crystal X-ray analysis.
A human subtelomeric repeat (designated as the HST repeat) has been isolated and characterized from a yeast artificial chromosome containing one human telomere. This repeat is located immediately adjacent to the telomeric T2AG3 repeats at the extreme termini of the human chromosomes. The DNA sequence of 3.6 kb of the HST repeat has been determined. The HST repeat spans over 3.6 kb in length, and contains one evolutionarily conserved CpG-rich region. The copy number of the HST repeat varies among telomeres. Genomic hybridization experiments suggest that the HST repeat consists of two distinct segments, and the distal portions of the HST repeat are also distributed elsewhere in the genome. In HeLa cells, the HST repeat sequence appears to be transcribed into a 6 kb polyadenylated RNA and a variety of non-polyadenylated RNA species.
C repeats constitute the predominant family of short interspersed repeats (SINEs) in the rabbit genome. Determination of the nucleotide sequence 5' to rabbit zeta-globin genes reveals clusters of C repeats, and analysis of these and other sequenced regions of rabbit chromosomes shows that the C repeats have a strong tendency to insert within or in close proximity to other C repeats. An alignment of 44 members of the C repeat family shows that they are composites of different sequences, including a tRNA-like sequence, a conserved central core, a stretch of repeating CT dinucleotides, and an A-rich tract. Cladograms generated by both parsimony and cluster analysis subdivide the C repeats into at least three distinct subfamilies. Nucleotides at sites diagnostic for subfamilies appear to have changed in a punctuated and progressive manner during evolution, indicating that a limited number of progenitors have given rise to new repeats in waves of dispersion. C repeats that insert into preexisting C repeats belong to subfamilies that are proposed to have been propagated more recently; hence, these data support the model of dispersion in successive waves. The divergence among the oldest group of C repeats is greater than that observed for the analogous Alu repeats in humans, indicating that rabbit C repeats have been propagating longer than human Alu repeats. The improved consensus sequence for these repeats is similar to that of the predominant artiodactyl SINE in both the tRNA-like region and a central region. Because members of different subfamilies cross-hybridize very poorly, hybridization data with representatives of each subfamily provide a new minimal estimate, 234,000, for the copy number of C repeats in the rabbit haploid genome, although it is likely that the actual value is closer to 1 million.
Human telomeres have been successfully cloned in Saccharomyces cerevisiae by complementing deficient yeast artificial chromosomes (YACs). This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes. Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes. Here we describe the techniques that are currently being used in cloning of telomeric DNA.
A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.
The genetic defect responsible for Huntington disease was originally localized near the tip of the short arm of chromosome 4 by genetic linkage to the locus D4S10. Several markers closer to Huntington disease have since been isolated, but these all appear to be proximal to the defect. A physical map that extends from the most distal of these loci, D4S90, to the telomere of chromosome 4 was constructed. This map identifies at least two CpG islands as markers for Huntington disease candidate genes and places the most likely location of the Huntington disease defect remarkably close (within 325 kilobases) to the telomere.
The addition of two embryonic globin genes, zeta 0 and zeta 4, to the rabbit alpha-like globin gene cluster expands it to include eight genes arranged 5'-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-zeta 4-3'. The identification of these new genes supports the model that this gene cluster evolved by a series of block duplications of gene sets. The nucleotide sequence of three embryonic zeta-globin genes, zeta 1, zeta 2, and zeta 3, shows that all three are pseudogenes. Gene zeta 1 contains two frame-shift deletions, gene zeta 2 has lost exon 1 as well as the 5' promoter sequences, and gene zeta 3 has lost and replaced codons for amino acids that are critical for the function of alpha- and zeta-globin polypeptides. However, genes zeta 1 and zeta 3 are still transcriptionally competent, as shown by the accurate initiation and processing of transcripts from cloned genes introduced into HeLa cells. A quantitative comparison of the zeta-globin gene sequences indicates that the ancestor to the rabbit zeta 1 and zeta 3 genes was inactivated about 44 million years ago, and the block duplication that formed the two genes occurred about 28 million years ago. About 600 base pairs of the 5'-flanking sequence of the rabbit zeta 3-globin gene is very similar to the 5' flanks of three other mammalian zeta-globin genes.
The alpha-like globin gene cluster in rabbits contains embryonic zeta-globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.
In order to understand the coordinate regulation between the alpha-like and beta-like globins during the developmental switches in hemoglobin synthesis, we have studied the rabbit alpha-like globin gene family. A cluster of six linked genes arranged 5'-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3' has been isolated as a set of overlapping clones from a library of rabbit genomic DNA. Blot-hybridization analysis of genomic DNA not only confirms this linkage arrangement but also reveals the presence of additional zeta and theta genes. We propose that this gene cluster was generated by a block duplication of a set of alpha-like genes; the proposed duplication unit is zeta-zeta-alpha-theta. Further duplications of a zeta-zeta-theta set are also proposed to have occurred. As expected for a duplicated locus, the rabbit alpha-like gene cluster contains long blocks of internal homology. The Z homology block is about 7.2 kilobase pairs long and contains the zeta genes; the T homology block is about 4.7 kilobase pairs long and contains a theta gene. Surprisingly, both Z and T homology blocks are flanked by a common junction sequence (J) which contains a region very similar to the 3'-untranslated sequence of an alpha-globin gene. Analysis of the J sequences suggests a recombination mechanism by which the alpha gene could have been deleted from the second set of genes in the cluster (zeta 2-zeta 3-theta 2). The relationships among the genes in characterized alpha-like gene clusters in mammals are summarized. The rabbit gene cluster differs from those of other mammals principally in the loss of a gene orthologous to the human psi alpha 1 and in the block duplication of the zeta-zeta-alpha-theta gene set.
Explore the source record for details and available documents.
The sequence of the DNA between two pseudogenes in the human alpha-like globin gene cluster has been determined. Comparison of this sequence with sequences from other alpha-like globin gene clusters revealed another pseudogene, psi alpha 2, between the previously recognized pseudogenes zeta 1 and psi alpha 1. Therefore, the human alpha-like globin gene family is organized 5'-zeta 2-zeta 1-psi alpha 2-psi alpha 1-alpha 2-alpha 1-3'. The new pseudogene psi alpha 2 is very close to zeta 1, beginning only 65 base pairs 3' to the polyadenylation site of zeta 1. The first exon and the first intron of psi alpha 2 are interrupted by large inserts which are flanked by short (6 to 8 base pairs) direct repeats. The pseudogene psi alpha 2 lacks a promoter for transcription by RNA polymerase II, the first exon is highly divergent, one splice site is mutated, and five different frameshift mutations have occurred in the coding regions. Thus psi alpha 2 cannot encode a globin polypeptide. This pseudogene was not recognized in previous hybridization analyses of the human alpha-like globin gene cluster, and our discovery of it by sequence analysis suggests that divergent copies of a large number of genes may comprise a substantial fraction of the slowly renaturing DNA of mammalian genomes.
A cloned 13.3-kilobase (kb) region of rabbit genomic DNA contains a cluster of alpha-like globin genes arranged 5'-psi zeta-(3.6 kb)-alpha 1-(2.2 kb)-psi alpha-3'. Genomic blot hybridization data show that this is the major alpha-like globin gene cluster in rabbits, although a second alpha-globin gene (alpha 2) is also detected. Repetitive sequences from the C family of short repeats flank the psi zeta gene, and a new repetitive element, the F repeat is located 3' to psi alpha. The sequence was determined for a 4024-base pair (bp) segment that extends from 149 bp 5' to the cap site of alpha 1 to 207 bp 3' to psi alpha. Gene alpha 1 is functional and encodes one of the major allelic variants of rabbit alpha-globin. This gene has very short introns (77 bp in intron 1 and 83 bp in intron 2) and an unusual ATA box in the 5' flanking region (CTTAAA), which does function to promote transcription by RNA polymerase II in a cell-free system. Short (4 or 9 bp) G + C-rich repeats are interspersed throughout the flanking regions and the introns. Gene psi alpha cannot encode a globin polypeptide, and it is probably inactive. This is shown by the absence of a normal globin gene promoter, the replacement of the 5' untranslated sequence by tandem repeats of the sequence GCCCGCCGC, frameshift deletions in exon 2, and the modification of the polyadenylation signal to AGTAAA. The intergenic region between alpha 1 and psi alpha is very G + C rich (65.7% G + C) and contains many short, tandem repeats. Gene psi zeta hybridizes specifically to a human zeta-globin gene probe, but a partial sequence reveals frameshift mutations that probably make psi zeta a pseudogene. Mammalian alpha-globin gene clusters vary in the presence or absence of pseudogenes, and if present, the position of the pseudogene differs in various gene clusters. Among the mammalian alpha-like gene clusters so far analyzed, the rabbit gene cluster is unique in the absence of duplicated alpha-globin genes that are undergoing concerted evolution.
The C repeat family was first observed in the rabbit beta-like gene family. We have estimated the repetition frequency of the C repeats, determined the nucleotide sequence of three intact members and one truncated member, and have investigated the size, tissue specificity, and intracellular localization of C repeat transcripts. Members of the C repeat family are short (average size of 316 base-pairs) and are repeated about 170,000 times per haploid genome in a widely dispersed pattern. They end in a 3' poly(dA) tract and are flanked by direct repeats that range in size from 8 to 16 base-pairs. The consensus internal control regions for polymerase III transcription are located near the 5' end. Different amounts of C repeat RNA accumulate in a variety of tissues, and most of the transcripts are confined to the nucleus. A heterogeneous distribution of C repeat RNA sizes was found, ranging from about 330 to 8200 nucleotides. These structural and transcriptional properties are similar to those of primate and rodent Alu and Alu-like repeats. However, the C repeats are not similar in sequence to the Alu repeats. Thus two different types of short, interspersed repeats capable of being transcribed and proposed to be transposable elements have now been identified in mammals. The positions of these short repeats in mammalian beta-like globin gene families are not tightly conserved.
Explore the source record for details and available documents.