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C K Shen

Publications and source records attributed to C K Shen.

At least 55 records · Page 3Linked to original sources
PubMed1986

A previously undetected pseudogene in the human alpha globin gene cluster.

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.

Base Sequence↗
PubMed1986

The orangutan adult alpha-globin gene locus: duplicated functional genes and a newly detected member of the primate alpha-globin gene family.

We have cloned and sequenced the complete alpha 1- and alpha 2-globin genes of the orangutan, and here we compare them to the homologous genes of the human. The pattern of similarity apparent among the genes is most consistent with a model of gene correction operating on the primate alpha-globin cluster. This correction breaks down in both human and orangutan in the 3'-untranslated region at 14 base pairs downstream from the termination codon. The unit evolutionary period values calculated for either the replacement substitution or the silent substitutions are only slightly higher than the previously established molecular clock predicts. The 7-base-pair insertion in intron 2 of the human alpha 1-globin gene is not present in either orangutan gene, suggesting that this insertion is not the cause of the sequence divergence in the 3'-untranslated regions of primate alpha 2- and alpha 1-globin genes. Finally, blotting hybridization and partial DNA sequencing reveal a newly detected member of the primate alpha-globin gene family, which is located downstream from the duplicated adult alpha-globin genes.

Animals↗
PubMed1986

DNA supercoiling of recombinant plasmids in mammalian cells.

We have used chloroquine/agarose gel electrophoresis and a blot-hybridization technique to study the modulation of superhelicity of extrachromosomal DNA in mammalian cells. The high sensitivity of the procedure has allowed us to measure the change in the specific linking difference or superhelical density (sigma) of a plasmid, psvo alpha 1p3d, after its introduction into COS-7 cells by DNA transfection. Because the molecular weight of psvo alpha 1p3d is approximately the same as that of simian virus 40 (SV40) DNA, the latter can be used as a standard for estimating the average linking difference or number of superhelical turns (tau) of psvo alpha 1p3d after separation of the different supercoiled species on chloroquine/agarose gels. It was found that transfection of monkey cells with either fully supercoiled psvo alpha 1p3d isolated from bacteria (tau = -27 +/- 1, sigma congruent to -0.051) or its relaxed form after treatment with DNA topoisomerase I yields psvo alpha 1p3d samples of the same tau and sigma values of -20 +/- 1 and -0.038, respectively. The difference between the tau values of psvo alpha 1p3d and SV40 in COS-7 cells, in which both plasmids undergo rounds of replication, corresponds to an average difference of 5 +/- 1 superhelical turns. Plasmid psvo alpha 1p3d remains at this lower level of superhelicity for at least 72 hr. The distribution in linking numbers of the topoisomers of psvo alpha 1p3d isolated from transfected COS cells is also more heterogeneous than that of SV40 DNA. These results suggest that the regulation of DNA supercoiling and chromatin assembly may be closely associated with specific DNA sequences. The approach presented here should have a wide application in the study of the regulation and functional role(s) of DNA supercoiling of plasmids in mammalian cells.

Animals↗
PubMed1986

Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat.

Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency.

Base Sequence↗
PubMed1985

Characterization of an unique RNA initiated immediately upstream from human alpha 1 globin gene in vivo and in vitro: polymerase II-dependence, tissue specificity, and subcellular location.

We have identified an abundant transcript initiated upstream from the canonical cap site of human alpha 1 globin gene in bone marrow cells and in COS-7 cells transfected with an alpha 1 globin gene-containing plasmid. Similar to the major alpha 1 globin transcript, this upstream RNA is present almost exclusively in the cytoplasm of the transfected COS-7 cells. It is also synthesized efficiently in vitro by RNA polymerase II in the nuclear extracts prepared from a Hela cell line and an erythroleukemia cell line, K562. RNAs isolated from these cell lines, however, do not contain this upstream transcript. The putative 5' end of the alpha 1 globin upstream RNA is mapped by primer extension to base -45, which is located in between the CCAAT and TATA boxes. The synthesis of this RNA in vitro and in vivo, and the close proximity of its 5' end to the promoter of the alpha 1 globin gene suggest a common mechanism regulating the transcriptional initiation of both the upstream and the major alpha 1 globin RNAs.

Animals↗
PubMed1985

End-to-end transcription of an Alu family repeat. A new type of polymerase-III-dependent terminator and its evolutionary implication.

Four or more consecutive thymidine residues on the non-template strand and G + C-richness of flanking DNA are the two necessary characteristics of efficient RNA polymerase-III-dependent transcriptional terminators. We have identified, from the study of in vitro transcription of a human Alu family repeat, a new type of RNA polymerase-III-dependent transcriptional terminator. A 258 base-pair Alu repeat located on the 3' side of the human alpha 1 globin gene can be transcribed in a HeLa S-100 extract to generate three RNA species of lengths 404 to 408, 252 to 255 and 173 to 174 nucleotides, respectively. Kinetics, pulse-chase and RNA incubation experiments showed no significant internal processing of the longer transcripts into shorter ones. These data plus detailed RNA mapping demonstrated conclusively that the multiple Alu RNA species resulted from accurate initiation at the first base (5' end) of the repeat, and multiple termination downstream. The 3' end(s) of the major transcript (252 to 255 nucleotides) maps at the 3' end of the Alu repeat sequence where there are not four or more consecutive thymidine residues on the non-template strand. The functional domain of the terminator has been mapped to a 45 base-pair segment that includes 36 base-pairs of the 3' end sequence of the Alu repeat plus nine base-pairs downstream. The high efficiency of termination (greater than 90%), the lack of consecutive T residues, the richness in A + T content, and the proposed ability of the RNA to form an imperfect hairpin structure in the 3' region of the transcript, thus identify a new type of eukaryotic class III terminator. We compare the structure of this class III terminator with that of the bacterial rho-dependent terminator. We also discuss its implication in the mechanism(s) of amplification and dispersion of Alu sequences in the primate genomes.

Base Sequence↗
PubMed1985

Comparison of human and chimpanzee zeta 1 globin genes.

The DNA base sequences of the entire chimpanzee zeta 1 globin gene and an additional 1 kb of DNA flanking both the human and chimpanzee genes have been determined. Whereas the human zeta 1 gene contains a termination codon in the sixth position, the chimpanzee gene appears to be functional. This finding confirms Proudfoot et al.'s suggestion that the human zeta 1 gene was recently inactivated. Like the corresponding human zeta 1 and zeta 2 genes, the first and second introns of the chimpanzee zeta 1 gene are occupied largely by tandem repeats of short oligonucleotides. These tandem repeats have undergone several rearrangements since the divergence of the human and chimpanzee zeta 1 genes.

Amino Acid Sequence↗
PubMed1985

Evolution of Alu family repeats since the divergence of human and chimpanzee.

The DNA sequences of three members of the Alu family of repeated sequences located 5' to the chimpanzee alpha 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human.

Animals↗
PubMed1984

A gradient of sequence divergence in the human adult alpha-globin duplication units.

The nucleotide sequences of the two 5'-homology blocks of human alpha-globin gene duplication units were determined. The sequence difference between the two blocks is essentially zero in the 5' portions, and increases gradually toward the 3' ends until it reaches a value of 18 percent. This gradient of sequence divergence is similar to the distribution of the frequencies of gene conversion along several loci in Ascobolus and yeast. Hot spots for initiation of gene correction processes appear to exist near the 5' ends of the human alpha-globin duplication units. The data provide the physical evidence for polar gene correction process in a mammalian genome.

Base Sequence↗
PubMed1984

Distinctive sequence organization and functional programming of an Alu repeat promoter.

Plasmid clones containing a human Alu family repeat can be transcribed efficiently by RNA polymerase III in HeLa cell extract. This generated three RNA species, all of which initiated from the first base (+1) of the repeat. By studying the transcriptional properties of deletion clones, subclones, and topologically different DNA templates, we demonstrated that: supercoiled DNA templates are transcribed 3- to 5-fold more efficiently than are linear or nicked circular DNA molecules; a contiguous DNA helix in the transcription complexes that extends into the 5' flanking region of positions -30 to -85 is absolutely required for initiation to occur (this interaction does not involve recognition of specific DNA sequences); and similar to the adenovirus VAI RNA and tRNA genes, the Alu repeat 3' to the alpha 1-globin gene (designated 3'-alpha 1 Alu) contains a split intragenic promoter: an anterior element (positions +4 to +37) and a posterior element (positions +70 to +82). However, the promoter of the Alu repeat functions in distinctive ways in comparison to those of other RNA polymerase III-dependent genes. The posterior promoter element alone is sufficient and necessary for an accurate initiation to occur. The presence of the anterior promoter element, which by itself does not initiate transcription, enhances the transcriptional efficiency by a factor of 10- to 20-fold. Furthermore, the distance between the initiation sites and the posterior promoter element, but not the anterior promoter element, remains constant. These results suggest that the promoter of this Alu family repeat consists of at least two functionally different domains: a "directing element" (the posterior promoter element) that determines the accuracy of initiation and an "enhancing element" (the anterior promoter element) that is mainly responsible for the transcriptional efficiency.

Chromosome Deletion↗
PubMed1983

Superhelicity induces hypersensitivity of a human polypyrimidine . polypurine DNA sequence in the human alpha 2-alpha 1 globin intergenic region to S1 nuclease digestion--high resolution mapping of the clustered cleavage sites.

Supercoiled recombinant DNAs containing the human adult alpha-globin gene region have been probed with nuclease S1 in vitro. While agarose gel electrophoresis showed only one predominant, double-stranded cleavage generated by S1 within 6 kb of human DNA and 4 kb of pBR322 sequence, a high resolution gel analysis reveals that the unique S1-hypersensitive locus in the human adult alpha-globin gene region actually contains more than 15 authentic S1 cleavage sites closely spaced together. The mapping approach used here locates the specific S1 cleavage sites on both DNA strands at the nucleotide sequence level. Interestingly, most of these sites are mapped within a 90 bp stretch of GC-rich (66%) polypyrimidine . polypurine DNA that is located 1060 to 1150 bp upstream from alpha 1-globin gene. These results provide the first high resolution map of double-stranded S1-cleavage sites induced within a specific DNA sequence under supercoil strain. The distribution and relative cutting frequencies of these sites mapped are consistent with a slippage mechanism in which the simple repeating sequences are organized into base-mismatched duplex on supercoiled DNA.

Base Composition↗
PubMed1983

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.

Animals↗
PubMed1983

Molecular evolution of the human adult alpha-globin-like gene region: insertion and deletion of Alu family repeats and non-Alu DNA sequences.

Previous heteroduplex studies have revealed extensive sequence homology between the two human adult alpha-globin-like genes (alpha 2 and alpha 1) and their flanking regions. These homologous regions, which are interrupted by two blocks of nonhomology, each span approximately 4 kilobases [Lauer, J., Shen, C.-K. J. & Maniatis, T. (1980) Cell 20, 119-130]. We have determined 3 kilobases of DNA sequences within and flanking the nonhomologous blocks of these two tandem duplication units. A total of three Alu family repeats has been identified. Two of them are approximately 300 base pairs long and define the 3' ends of the first homology blocks. The third Alu family member is a 600-base-pair-long sequence consisting of two monomeric Alu members arranged in a head-to-tail fashion. It is located in the 3' portion of the first block of nonhomology in alpha 2-gene-containing unit. We present direct evidence that this dimeric Alu sequence was inserted at a staggered break. The second nonhomology block is the result of insertion or deletion of a 224-base-pair sequence. From these data and the calculation of sequence divergence, we propose a history for the evolution of the human adult alpha-globin-like gene region. We also suggest that DNA insertion elements may disrupt gene correction processes in the two duplication units containing alpha 2- and alpha 1-globin genes.

Adult↗
PubMed1982

The organization, structure, and in vitro transcription of Alu family RNA polymerase III transcription units in the human alpha-like globin gene cluster: precipitation of in vitro transcripts by lupus anti-La antibodies.

We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.

Antibodies↗