Analysis of the regions flanking the human insulin gene and sequence of an Alu family member

Bell, Graeme I.; Pictet, Raymond; Rutter, William J.

doi:10.1093/nar/8.18.4091

Cited by 147 publications

(82 citation statements)

References 32 publications

(37 reference statements)

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“…At 48 h post-VOL. 5,1985 on April 4, 2019 by guest http://mcb.asm.org/ Downloaded from transfection, total RNA was isolated from transfected cells maintained at 37°C or heat shocked for 2 h at 43°C. The RNAs were hybridized with pHBCAT and 5' end-labeled at the EcoRI sites, and the RNA-DNA hybrids were subsequently digested with Si nuclease.…”

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confidence: 99%

Structure and expression of the human gene encoding major heat shock protein HSP70.

Wu¹,

1985

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We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43°C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to Human tissue culture cells respond to heat shock, and certain other stimuli, by the induced synthesis of a small set of proteins (molecular weights 100,000, 70,000, and 37,000 [58]). The effect of heat shock on the pattern of protein synthesis in human cells is similar to that for other eucaryotic cells (reviewed in reference 55). This highly conserved response-the activation of a small number of genes and the repression of other normally active genes-has been most intensively studied in Drosophila (1). The altered protein synthetic pattern is, in general, a reflection of both the preferential transcription of heat shock genes and the selected translation of their mRNAs.The major heat shock protein synthesized by eucaryotic cells belongs to a family of 70,000-dalton proteins (HSP70). The conservation of HSP70 among species is revealed by the similar sizes, apparent isoelectric points, and tryptic peptide patterns (64). Indeed, polyclonal antibodies raised against chicken HSP70 cross-react with proteins of similar size from yeast, Drosophila, Xenopus, mice, and humans (32). In Drosophila, the HSP70 multigene family encodes two heat shock proteins, HSP68 and HSP70 (25), and three cognate proteins (27) whose synthesis occurs at normal temperature and which are not heat shock inducible. Drosophila melanogaster has five copies of the HSP70 gene, two at the 87A chromosomal locus and three at the 87C locus (25). Similarly, Saccharomyces cerevisiae contains two copies of the HSP70 gene (28).The sequence conservation of the HSP70 genes among species has been used to isolate the homologous genes from yeast (28) (31). In this study, we describe the structural features of the human HSP70 gene: the organization of the genomic clone and the location of the 5' and 3' termini of the heat shock-induced HSP70 transcript. We examine the expression of the human HSP70 gene in hamster cells and of a chimeric HSP70 bacterial chloramphenicol acetyltransferase (CAT) gene in human cells. MATERIALS AND METHODSGeneral methods. The human genomic lambda library (39) was generously provided by T. Maniatis. Approximately 5 x 105 recombinant phage were screened (22) for sequences homologous to a subclone (plasmid 232.1; 41) containing 1.1 kilobase (kb) of coding sequence adjacent to the 5' end of the Drosophila HSP70 gene. Hybridizing plaques were purified, and DNA was isolated from phage particles banded by equilibrium CsCl centrifugation (42).Genomic DNA was prepared from human placental tissue lysed with sodium dodecyl sulfate (SDS) and digested with proteinase K (8). Subclones of H3-1 were constructed with the v...

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Structure and expression of the human gene encoding major heat shock protein HSP70.

Wu¹,

1985

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“…The polymorphisms are revealed as variations in the length of the fragments produced by cleavage with restriction enzymes. They can be detected by the method of Southern (2), using as probes cloned arbitrary single-copy sequences (3) or specific genes [e.g., hemoglobin (4,5) and insulin (6)(7)(8)]. Several hundred evenly spaced genetic markers should be sufficient to map the entire human genome (9,10).…”

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Construction of a map of the short arm of human chromosome 6.

Leach¹,

DeMars²,

Hasstedt³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Four DNA sequences that reveal restriction fragment length polymorphisms (RFLPs) on the short arm of human chromosome 6 have been identified. Two of these sequences were isolated from recombinant DNA libraries enriched for DNA from human chromosome 6, one was isolated as a subclone from a human DNA segment having homology to an HLA class I sequence, and one was isolated by virtue of its homology to part of the insulin gene.

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“…These 2 homologous regions overlap 40 bp stretches (starting at residues 78 and 212, respectively) that have been observed to be highly conserved in human Alus and in rodent interspersed repeated sequence (36)(37)(38). The A residues at the end of the monkey sequence are preceded by a repetitive oligonucleotide AGAGAGAG that is complementary to the sequence TCTC found at a similar position in several human Alu sequences (18,37,39,40). We do not know whether this is a general feature of the African green monkey Alu family.…”

Section: Discussionmentioning

confidence: 99%

“…We do not know whether this is a general feature of the African green monkey Alu family. Like at least some human Alu sequences (37,39,40), the monkey Alu sequence is surrounded by short direct repeats. The repeated octanucleotide GCTTTGAG that flanks the monkey sequence differs both in sequence and length from the repeats flanking the human sequences, which also differ from one another (37,39,40).…”

Section: Discussionmentioning

confidence: 99%

“…Like at least some human Alu sequences (37,39,40), the monkey Alu sequence is surrounded by short direct repeats. The repeated octanucleotide GCTTTGAG that flanks the monkey sequence differs both in sequence and length from the repeats flanking the human sequences, which also differ from one another (37,39,40). The direct repeats of non-Alu sequence that flank Alu segments are reminiscent of the duplicated target sites around transposable elements (reviewed in reference 41).…”

Section: Discussionmentioning

confidence: 99%

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Interspersed repeated sequences in the African green monkey genome that are homologous to the human Alu family

1981

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The dominant family of interspersed repetitive DNA sequences in the human genome has been termed the Alu family. We have found that more than 75% of the X phage in a recombinant library representing an African green monkey genome hybridize with a human Alu sequence under stringent conditions. A group of clones selected from the monkey library with probes other than the Alu sequence were analyzed for the presence and distribution of Alu family sequences. The analyses confirm the abundance of Alu sequences and demonstrate that more than one repeat unit is present in some phages. In the clones studied, the Alu units are separated by an average of 8 kilobase pairs of unrelated sequences. The nucleotide sequence of one monkey Alu sequence is reported and shown to resemble the human Alu sequences closely. Hence, the sequence, dispersion pattern, and copy number of the Alu family members are very similar in the African green monkey and human genomes.Among the clones investigated were two that contain segments of the satellite DNA termed o-component joined to non o-component DNA. The experiments indicate that in the monkey genome Alu sequences can occur close to regions of a-component DNA.

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Analysis of the regions flanking the human insulin gene and sequence of an Alu family member

Cited by 147 publications

References 32 publications

Structure and expression of the human gene encoding major heat shock protein HSP70.

Structure and expression of the human gene encoding major heat shock protein HSP70.

Construction of a map of the short arm of human chromosome 6.

Interspersed repeated sequences in the African green monkey genome that are homologous to the human Alu family

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