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Biomedical subjects

E A Craig

Publications and source records attributed to E A Craig.

At least 127 records · Page 7Linked to original sources
PubMed1981

Cloned complementary deoxyribonucleic acid of Drosophila cells. Relationship of genome copy number to messenger ribonucleic acid abundance.

Recombinant DNA plasmids containing DNA sequence complementary to poly(adenylic acid) [(poly(A)] containing RNA from the cytoplasm of Drosophila Kc tissue culture cells were constructed. The reiteration frequency in the genome of the RNA homologous to the 20 randomly selected clones was determined by two rapid methods. Of the 20, 17 were determined to be single copy, 2 were repeated several (2-4) times, and 1 was repeated approximately 10 times. The steady-state level of mRNAs homologous to the 20 cDNAs was quantitated and varied more than 160-fold. The RNAs ranged from 0.16% to less than 0.001% of the poly(A)-containing RNA.

Animals↗
PubMed1981

Primary sequence of the 5' flanking regions of the Drosophila heat shock genes in chromosome subdivision 67B.

The 5' flanking regions of the four small heat shock genes of Drosophila melanogaster from cytological locus 67B have been characterized. Approximately 500 bp of the primary sequence upstream from the proposed site of initiation of translation has been determined and the 5' end of the messenger RNAs have been localized for each gene. Each of the four genes contains an A-T rich sequence, either TATAAATA or TATAAAAG, which is flanked by a G-C rich region. This A-T rich sequence, which ends about 23 bp upstream from the proposed site of initiation of transcription, is similar to those found in most eukaryotic genes. Novel features of these four genes include a region of homology beginning near the proposed site of initiation of transcription and extending about 20 bp into the 5' noncoding region of the genes. This sequence is also found in the gene for the major heat shock protein, hsp 70. The leaders of these five heat shock genes are long, from 111 to 253 bases in length, as well as unusually A rich, from 46% to 51% A. In addition, each of the four small genes contains the sequence ACTTTNA, 195 +/- 12 bp from the proposed site of initiation of transcription.

Animals↗
PubMed1980

Four Drosophila heat shock genes at 67B: characterization of recombinant plasmids.

A 33 kilobase region of the 67B locus of Drosophila melanogaster genome has been isolated. The genes for the 27K, 26K, 23K and 22K heat shock induced proteins are contained within an 11 kb segment. the 27K gene, approximately 1.4 kb in length, and the 23K gene, approximately 1.0 kb in length, are separated by about 1.5 kb of spacer DNA; a third block of transcription, which encodes the 26K gene, is approximately 1.1 kb in length, and is separated from the gene for the 23K protein by 4.5 kb of spacer DNA. The 22K gene is located approximately 1.2 kb from the 26K gene. The RNAs encoding the 27K, 23K and 22K genes are transcribed from the same DNA strand. With the assignment of the four small heat shock induced proteins to 67B, genes for all seven major heat shock induced proteins have no been mapped cytologically.

Animals↗
PubMed1980

Multiple actin-related sequences in the Drosophila melanogaster genome.

In this paper we describe the isolation and characterization of a 7.2 kb D. melanogaster chromosomal DNA fragment (K1) which contains nucleotide sequences complementary to D. melanogaster actin mRNA. Plasmid K1 was identified using a Dictyostelium actin cDNA plasmid, B1, as a probe. D. melanogaster mRNA selected by hybridization with immobilized K1 DNA was translated in vitro to yield products which co-migrate with the D. melanogaster actins I, II and III in two-dimensional gel electrophoresis and bind to DNAase I agarose. A physical map localizing restriction endonuclease cleavage sites in the K1 DNA fragment and the direction of transcription is presented. The position of the coding region has been localized by hybridization with labeled B1 DNA and with labeled poly(A)-containing D. melanogaster RNA. On the basis of hybridization of labeled subfragments of plasmid K1 to restriction endonuclease-cleaved D. melanogaster embryo DNA, we conclude that the nucleotide sequence of the presumptive coding region is responsible for labeling of a pattern of multiple restriction fragments from embryo DNA. The chromosomal locus from which DNA fragment K1 is derived has been localized by in situ hybridization to two closely linked bands in the region 88F. Related DNA sequences corresponding to putative actin genes have also been mapped cytologically. These results support the hypothesis that the genes for actin in D. melanogaster are members of a closely related family of coding sequences.

Actins↗
PubMed1980

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions.

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.

Amino Acid Sequence↗
PubMed1980

Genes for three Drosophila heat-shock-induced proteins at a single locus.

Two plasmids containing cDNA inserts complementary to the messenger RNAs for the 26,000 and 23,000 molecular weight heat-shock-induced proteins from Drosophila melanogaster were identified. Both of these probes hybridized to a site of a major heat-shock-induced puff, 67B. Hybridization by the gel transfer technique with genome DNA that had been cleaved by a restriction endonuclease indicated that the two genes are closely linked. This expectation was validated through isolation of a DNA fragment generated by BamHI endonuclease containing genes for the 26,000 and 23,000 molecular weight heat-shock-induced proteins and also a gene encoding the 27,000 molecular weight heat-shock-induced protein.

Animals↗
PubMed1979

Rapid quantitation of individual RNA species in a complex population.

Many investigations require quantitation of one or more individual RNA species in complex populations. Existing methods are tedious when multiple samples are to be assayed. A method is presented which allows rapid and accurate quantitation of many species of RNA simultaneously. Recombinant plasmids containing cDNA inserts are electrophoresed in agarose and blotted to nitrocellulose. After hybridization with labeled RNA and autoradiography, bands are quantitated by scanning. The results were calibrated by solution hybridization. The approach has been validated through the use of plasmids containing inserts of Drosophila cDNA and RNA of cultured cells.

Animals↗
PubMed1979

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.

Animals↗
PubMed1976

Nuclear transcripts larger than the cytoplasmic mRNAs are specified by segments of the adenovirus genome coding for early functions.

Nuclear viral RNAs synthesized early in productive infection with adenovirus 2 were analyzed by hybridization to specific viral DNA fragments. Radioactive RNAs were subjected to electrophoresis in polyacrylamide gels containing 98% formamide, and the fractionated RNA was hybridized to specific DNA fragments generated by cleavage with endo R-Eco Ri or endo R-Sma l. The viral genes expressed early in infection are located in four different segments of the genome. When nuclear RNA was hybridized to DNA fragments representing each of these segments, discrete RNA size classes were detected. For each of these four regions of the genome, some of the discrete nuclear RNAs were larger than the cytoplasmic mRNAs. As calculated from electrophoretic mobility, the molecular weight of these nuclear RNA size classes ranged from 15% to several fold greater than that of the corresponding cytoplasmic mRNAs. Hybridization-inhibition experiments were performed to analyze the sequences present in one of these larger nuclear transcripts, a 22S RNA transcribed from the left-hand end of the genome. This 22S nuclear RNA was transcribed from the same strand as the viral mRNA, for it contained cytoplasmic sequences; it also contained sequences restricted to the nucleus. The larger nuclear RNAs may serve as precursors to the cytoplasmic viral mRNAs synthesized early in infection.

Adenoviridae↗
PubMed1975

Sequence relationships between adenovirus 2 early RNA and viral RNA size classes synthesized at 18 hours after infection.

Synthesis of cytoplasmic viral RNA was studied during infection of cultured human (KB) cells with adenovirus 2. At 6 h, before viral DNA synthesis began 5% of the poly(A)-containing RNA hybridized to viral DNA; by 12 h and at later times more than 80% was virus specified. At 18 h after infection, four major size classes of cytoplasmic viral RNA were identified among the poly(A)-containing molecules. These size classes migrated as 27S, 24S, 19S, and 12 to 15S in polyacrylamide gels. The three larger size classes could also be identified in denaturing formamide gels. Hybridization of the 27S, 24S, and 19S viral RNAs was not inhibited by RNA harvested from cells at early times in infection. Therefore, these three major RNAs must code for late viral proteins. Hybridization of the 12 to 15S RNA was partially inhibited by RNA from cultures harvested at early times, suggesting that in this size class some of the RNA labeled at 18 h codes for early viral proteins.

Adenine Nucleotides↗
PubMed1975

Analysis of early adenovirus 2 RNA using Eco R-R1 viral DNA fragments.

Adenovirus 2 RNA synthesized early in productive infection was analyzed by RNA-DNA hybridization. Hybridization experiments were performed with adenovirus 2 DNA and wit, the six adenovirus 2 DNA fragments generated 0y digestion with the restriction endonuclease Eco R.R1. Duplex formation between RNA and -32P-labeled viral DNA was assayed by S(1) nuclease digestion. RNA from the cytoplasm annealed 12 percent of the total viral DNA and the following percentage of each of the R.R1 fragments: 6 percent of R1-A, 24 percent of R1-B, 0 percent of R1-F, 40 percent of R1-D, 13 percent of R1-E, and 22 percent of R1-C. The early cytoplasmic RNA is composed of two sequence classes: class I, present in greatly reduced quantities at late times in infection (18 h), and class II, which remains at high concentrations at 18 h. In hybridization-inhibition experiments, hybridization of class II RNA is inhibited by late cytoplasmic RNA, whereas hybridization of class I RNA is not blocked by late cytoplasmic RNA (J. J. Lucas and H. S. Ginsberg, 1971; E.A. Craig and H. J. Raskas, 1971). To determine the location of class I and II sequences on the genome, membrane bound DNA fragments were used in hybridization-inhibition experiments. These studies demonstrated that the early cytoplasmic transcripts of R1-D belong to class II, whereas R1-C transcripts are class I sequences. The cytoplasmic RNAs transcribed from fragments A and B contain both class I and class II sequences. Analysis of cytoplasmic RNA fractionated by size demonstrated that the class I sequences include a 19 S RNA transcribed from R1-B and class II sequences include a 20S RNA derived from R1-D. Nuclear RNA purified from cultures early in infection was annealed with -32P-labeled R1 fragments. With all six fragments the nuclear RNA annealed as much or more of the DNA than did cytoplasmic RNA. Eco R1-F annealed at least 25 percent with early nuclear RNA, whereas no sequences homologous to R1-F were detected in early cytoplasmic RNA. When cultures were labeled from 2 to 6 h after infection, at least 5 percent of the -3H-labeled early nuclear viral RNA annealed to Eco R1-F. Some of these nuclear transcripts from R1-F appear to be covalently linked to sequences transcribed from a contiguous region of the genome (Eco R1-B). 8.4 percent of the RNA selected by hybridization of R1-F reannealed to R1-B, whereas no more than 1.5 percent reannealed to R1 fragments A, D, E, or C.

Adenoviridae↗
PubMed1975

Identification of early adenovirus type 2 RNA species transcribed from the left-hand end of the genome.

Unique fragments of adenovirus type 2 DNA generated by cleavage with endonuclease R-Eco RI or endonuclease R-Hsu I (Hin dIII) were used to map cytoplasmic viral RNAs transcribed early in productive infection. Radioactive early viral RNA was first fractionated by polyacrylamide gel electrophoresis. Eluted viral RNAs were then tested for hybrid formation with DNA fragments. The Eco RI DNA fragment (Eco RI-A) which contains the left-hand 58% of the genome hybridized 13S and 11S RNAs. More detailed mapping of these RNAs was achieved by hybridization to the seven Hsu I fragments of Eco RI-A. The early RNA annealed only to Hsu I-G and C, two fragments which comprise the extreme left-hand 17% of the genome. Viral RNA migrating as 13S and 11S annealed to Hsu I-G, and 13S RNA annealed to Hsu I-C. A 13S RNA is transcribed from Eco RI-A late in infection (18 h). Hybridization-inhibition studies with Eco RI-A DNA, early cytoplasmic RNA, and 3H-labeled 13S late RNA demonstrated that this RNA synthesized at late times is an early RNA species which continues to be synthesized in large amounts at 18 h. This 13S RNA synthesized at 18 h hybridized to Hsu I-C but not to Hsu I-G DNA. These results establish that the 13S RNAs transcribed from Hsu I-G and C at early times must be different species.

Adenoviridae↗
PubMed1974

Localization of adenovirus 2 messenger RNA's to segments of the viral genome defined by endonuclease R-R1.

Adenovirus 2 mRNAs synthesized in productive infection were assigned to specific regions of the genome by hybridization to unique fragments of viral DNA. Radioactive viral RNA synthesized early or late in infection was first fractionated by polyacrylamide gel electrophoresis. Eluted RNAs were then tested for complementary hybrid formation with each of the six fragments of adenovirus 2 DNA generated by cleavage with endonuclease R.R1. Early RNA species migrating as 13S, 19S, and 20S RNAs were identified as transcription products of fragments A, B, and D, respectively. In addition to the 13S RNA transcribed from A fragment DNA, 13S RNA also hybridized to the D and E fragment DNAs; 11S RNA annealed to both A and B fragments. The RNA that hybridized to fragment C DNA was heterogeneous in size. Viral RNA synthesized late in infection included 27S, 24S, 19S, and 11S size classes, all of which annealed to A fragment DNA. Additional RNA migrating as 24S hybridized to E and C fragment DNA, and 23S RNA annealed to F fragment DNA. The results of the hybridizations of size fractionated RNAs with viral DNA fragments enabled formation of a partial map of viral mRNAs with respect to the adenovirus 2 genome. Some of the viral RNAs may represent transcripts which overlap R1 cleavage sites, because in at least three instances hybridization revealed viral RNAs which have the same electrophoretic mobility and anneal to fragments that are contiguous on the genome.

Adenoviridae↗