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C K Shen

Publications and source records attributed to C K Shen.

70 records · Page 4Linked to original sources
PubMed1980

The chromosomal arrangement of human alpha-like globin genes: sequence homology and alpha-globin gene deletions.

We report the isolation of a cluster of four alpha-like globin genes from a bacteriophage lambda library of human DNA (Lawn et al., 1978). Analysis of the cloned DNA confirms the linkage arrangement of the two adult alpha-globin genes (alpha 1 and alpha 2) previously derived from genomic blotting experiments (Orkin, 1978) and identifies two additional closely linked alpha-like genes. The nucleotide sequence of a portion of each of these alpha-like genes was determined. One of these sequences is tentatively identified as an embryonic zeta-globin gene (zeta 1) by comparison with structural data derived from purified zeta-globin protein (J. Clegg, personal communication), while the other sequence cannot be matched with any known alpha-like polypeptide sequence (we designate this sequence phi alpha 1). Localization of the four alpha-like sequences on a restriction map of the gene cluster indicates that the genes have the same transcriptional orientation and are arranged in the order 5'-zeta 1-phi alpha 1-alpha 2-alpha 1-3'. Genomic blotting experiments identified a second, nonallelic zeta-like globin gene (phi 2) located 10-12 kb 5' to the cloned zeta-globin gene. Comparison of the locations of restriction sites within alpha 1 and alpha 2 and heteroduplex studies reveal extensive sequence homology within and flanking the two genes. The homologous sequences, which are interrupted by two blocks of nonhomology, span a region of approximately 4 kb. This extensive sequence homology between two genes which are thought to be the products of an ancient duplication event suggests the existence of a mechanism for sequence matching during evolution. One consequence of this arrangement of homologous sequences is the occurrence of two types of deletions in recombinant phage DNA during propagation in E. coli. The locations and sizes of the two types of deletions are indistinguishable from those of the two types of deletions associated with alpha-thalassemia 2 (Embury et al., 1979; Orkin et al., 1979; S. Embury et al., manuscript submitted). This information strongly suggests that the genetic disease is a consequence of unequal crossing over between homologous sequences within and/or surrounding the two adult alpha-globin genes.

Bacteriophage lambda↗
PubMed1980

The organization of repetitive sequences in a cluster of rabbit beta-like globin genes.

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit beta-like globin genes (beta 1-beta 4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140-400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.

Animals↗
PubMed1980

Tissue-specific DNA methylation in a cluster of rabbit beta-like globin genes.

The relationship between DNA methylation and differential expression of rabbit beta-like globin genes was studied by using restriction enzymes that cleave the sequence C-C-G-G but are differentially inhibited by the presence of 5-methylcytosine. The methylation frequency of 13 C-C-G-G sites that flank a set of four closely linked rabbit beta-like globin genes was determined. This analysis revealed that certain sites surrounding embryonic and adult globin genes are relatively undermethylated in DNA from embryonic and adult erythroid tissues, respectively. This pattern is most pronounced for three sites that are undermethylated in erythroid cells but are totally methylated in nonerythroid cells. We conclude that the degree of CpG methylation in the rabbit beta-like globin gene cluster is correlated with gene activity, but the effect is confined to relatively small regions of DNA.

Animals↗
PubMed1978

Photochemical crosslinking of transcription complexes with psoralen. I. Covalent attachment of in vitro SV40 nascent RNA to its double-stranded DNA template.

14C-labeled SV40 DNA has been transcribed with E. coli RNA polymerase using 3H-labeled ribonucleotide triphosphates as precursors. The resulting transcription complexes were then photochemically crosslinked with the psoralen derivative, 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT), at 37 degrees C and analyzed in SDS-sucrose gradients. It was found that the photochemical crosslinking procedure caused the nascent RNA chains to co-sediment with their double-stranded (helical) SV40 templates in the denaturing sucrose gradient. This result and several control experiments suggest that covalent linkages have formed between nascent RNA and helical DNA after the photochemical reaction. The crosslinking phenomenon was observed to be independent of the superhelical state of the DNA used as the template. Prior addition of EDTA to stop the transcription is not required for successful crosslinkage.

Coumarins↗
PubMed1977

Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA.

The synthesis of five new psoralen derivatives is described. Three of these, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, 4'-methoxymethyl-4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, and characterized with respect to their photoreactivity with DNA and RNA. They are found to be greatly superior to 4,5',8-trimethylpsoralen and 8-methoxypsoralen, the two commonly used psoralens, in their abilities to saturate the photoreactive sites on DNA and RNA without repeated addition of reagent. A simplified mechanism for the photoreaction of psoralens with nucleic acids is presented and provides a basis for understanding the superior properties of these compounds. The compounds have superior reactivity not only with isolated DNA and RNA but also in viruses and in cells. Psoralens are shown for the first time to cross-link RNA double helices.

Binding Sites↗
PubMed1977

Mapping of sequences with 2-fold symmetry on the simian virus 40 genome: a photochemical crosslinking approach.

Sequences with 2-fold axes of symmetry have been detected and mapped on the simian virus 40 (SV40) genome by their ability to form hairpin turns in single-stranded SV40 DNA. Supercoiled SV40 DNA (SV40 I) was digested with restriction enzymes EcoRI and HpaII. The resulting linear DNA molecules with lengths of the complete SV40 genome were then denatured and photochemically reacted with 4,5',8-trimethylpsoralen (trioxsalen) at 16.0 +/- 0.5 degrees and different ionic strengths. Secondary structures on the single-stranded SV40 DNA were crosslinked and their positions analyzed by electron microscopy. There were no observable hairpin turns on the denatured SV40 DNA when it was photoreacted in 1 mM Tris-HCl/0.1 mM EDTA at pH 7.0. In 20 mM NaCl, one specific hairpin turn was detected at 0.17 +/- 0.02 map units on the map of EcoRI-digested SV40 DNA, where the 3' ends of early 19S mRNA, late 19S mRNA, and 16S mRNA of SV40 have been mapped. In 30 mM NaCl there are five more major hairpin turns besides the most stable one. The centers of four of these specific hairpin turns were mapped at 0.26 +/- 0.02, 0.68 +/- 0.03, 0.84 +/- 0.02, and 0.94 +/- 0.01 units on the map of EcoRI-digested SV40. The fifth one is at or near the unique EcoRI cleavage site on SV40 DNA. The possible functions of these sequences are discussed in terms of the nature of the promoter sites, the replication origin, the processing of RNA precursors, and regulation at the translational level.

Base Sequence↗
PubMed1976

Cross-linking of DNA in situ as a probe for chromatin structure.

The photochemical cross-linking of DNA in situ in chromatin is blocked over short intervals. Electron microscopy of DNA cross-linked in chromatin reveals the lengths of protected regions and provides a map of their sites along the DNA. Protected regions occur most frequently in tandem and have a basic length of 160 to 200 base pairs.

Animals↗
PubMed1976

Psoralen-crosslinked secondary structure map of single-stranded virus DNA.

The photochemical crosslinking of DNA by 4,5',8-trimethylpsoralen (trioxsalen) has been used to freeze the secondary structures of single-stranded DNA molecules of bacteriophage fd at different ionic strengths. These secondary structures (hairpins or looped hairpins) have been visualized in the electron microscope. Most of the single-stranded circular fd DNA molecules show only one hairpin after irradiation at 15 degrees in 20 mM NaCl in the presence of trioxsalen. As the ionic strength is increased, more hairpins appear on the DNA molecules. To map these secondary structures, double-stranded supercoiled fd DNA (RFI) was cleaved with the restriction enzyme HindII, which makes only one cut on each RFI molecule. After denaturation and crosslinking of the linear single-stranded fd DNA (a mixture of viral and complementary strands), all the hairpins have been mapped on the DNA molecule with respect to this HindII site. The results show that these hairpins occur at specific sites. The most stable hairpin has been assigned to the position where the initiation site for the conversion of single-stranded fd DNA to the double-stranded covalently closed form has been mapped. The remaining hairpins map in or near regions corresponding to in vitro promoter sites on the fd DNA.

Chromosome Mapping↗
PubMedDate not supplied

Sequence organization and genomic complexity of primate theta 1 globin gene, a novel alpha-globin-like gene.

The alpha-like and beta-like globin genes have provided a paradigm for the study of molecular evolution and regulation of multigene families in eukaryotes. The human alpha-globin gene cluster, which is on chromosome 16 (ref. 1), consists of six genes arranged in the order 5'-zeta(embryonic)-psi zeta-psi alpha 2-psi alpha 1-alpha 2(adult)-alpha 1(adult)-3'. DNA sequencing data have demonstrated that zeta (ref. 6) and alpha 2 (or alpha 1, refs 7-9) are the embryonic and adult genes, respectively, while psi zeta (ref. 6), psi alpha 2 (ref. 5) psi alpha 1 (ref. 10) are all inactive pseudogenes. Restriction mapping analysis has shown that the structure of this locus in several anthropoid primates is nearly identical to that of the human. Recently, we have isolated the adult alpha-globin gene region from orang-utan, olive baboon and rhesus macaque by molecular cloning. We report here the complete nucleotide sequence of a gene located immediately downstream from the adult alpha 1-globin gene of the orang-utan, along with its flanking DNA. We designate this gene as theta 1, and show that it contains the essential sequence elements required for an expressive gene. The putative polypeptide is 141 amino acids long, identical to that of the alpha- or zeta-globin, but its predicted amino-acid sequence is nearly as different from the orang-utan alpha-globin (55 differences) as the human zeta-globin is from the human alpha-globin (59 differences), suggesting an ancient history for the theta 1-globin gene. Results of blot hybridization experiments using the cloned orang-utan theta 1 gene sequence as probe demonstrate a similar alpha 2-alpha 1-theta 1 linkage map existing in the human genome. Furthermore, multiple copies of sequences homologous to the theta 1 gene are detected in both human and orang-utan. These results cast a new light on the primate alpha-globin gene family, and have intriguing implications for the existence of previously unreported, functional globin-like gene(s) in the primate genomes.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence that the recently discovered theta 1-globin gene is functional in higher primates.

A new subfamily of the alpha-globin-like family has recently been identified in higher primates, rabbit, galago and possibly the horse. One member of this subfamily, theta 1, is downstream from the adult alpha 1-globin gene. In orang-utan, but not in rabbit or galago, the theta 1-gene appears to be structurally intact, suggesting that it may be functional in this species. The orang-utan theta 1-gene possesses initiation and termination codons, and the predicted polypeptide differs from the orang-utan alpha 1-globin by 55 amino acids. The upstream promoter boxes CCAAT and ATA are present, although approximately 150 base pairs (bp) farther upstream than in the alpha 1-gene. This structural difference in the promoter between the orang-utan theta 1- and alpha 1-genes has led Proudfoot to speculate that the theta 1-gene may be inactive. We have now cloned the theta 1- and alpha 1-globin genes from the olive baboon, and have compared their sequences with those of orang-utan. The unique promoter structure of the orang-utan theta 1-gene is highly conserved in baboon, although the orang-utan and baboon diverged nearly 30 million years ago. The coding sequences of the two theta 1-genes differ by only 6.3% with 22 out of 27 nucleotide substitutions being codon third position silent changes. These data support the view that the theta 1-gene has been functional in the baboon, orang-utan, and by implication, in man. We also estimate that the duplication event generating the theta 1- and alpha-globin-like subfamilies may have occurred as much as 260 million years ago.

Amino Acid Sequence↗