Biomedical subjects
Publications and source records attributed to J Shine.
DNA complementary to mRNA coding for the human polypeptide hormone, chorionic somatomammotropin, has been purified by specific restriction endonuclease digestion and religation before cloning into bacterial plasmids. The primary structure of a major portion of this mRNA species is deduced from the nucleotide sequence of the recombinant DNA.
Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.
The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColE1 DNA as substrates. The reaction was not linearly dependent on enzyme concentration but increased markedly at high enzyme concentrations. Although T4 RNA ligase did not catalyze this blunt end joining, it makedly stimulated the DNA ligase reaction particularly at low DNA ligase concentrations. The apparent Km for the decamer was 50 micronM in the presence or absence of RNA ligase. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments were mostly linear oligomers but included some circular duplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction.
Explore the source record for details and available documents.
A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.
Transcription of DNA from the RNA genome of avian sarcoma virus by RNA-directed DNA polymerase in vitro initiates on a primer (tRNATrp) located near the 5'-terminus of the viral genome. One of the major products of transcription is a single-stranded DNA chain complementary to a sequence of 101 nucleotides immediately distal to the site of initiation of DNA synthesis. We have determined the complete nucleotide sequence of this transcribed chain for the Prague strain of avian sarcoma virus, a partial sequence of the transcribed chain for the Bratislava 77 strain of avian sarcoma virus, and the sequence of a DNA transcript that is shorter than the transcribed single-stranded chain. Our data define the location of tRNATrp on the genome of avian sarcoma virus and provide the sequence of 119 nucleotides at the 5'-terminus of the genome. Portions of this sequence may be involved in the binding of RNA-directed DNA polymerase, the initiation of translation from viral messenger RNA, the extension of RNA-directed DNA synthesis from the 5'- to the 3'-terminus of viral RNA, and the integration of viral DNA into the host genome.
In summary, a general approach is presented to purify and sequence DNA fragments of a specific gene starting with a heterogeneous mixture of mRNAs. The methodology has been applied to the determination of the DNA sequence of a portion of the gene for human chorionic somatomammotropin. Most of the possible translation codons of the genetic code were found to be used. Some selectivity in the codon choices was found, and this may be important for RNA or gene regulation or structure. The stop codon UAG was found and a second stop codon in the same reading frame was found nine bases farther down. Finally, a "palindrome" sequence was detected in the 3' noncoding region.
Explore the source record for details and available documents.
We have examined the 3'-terminal sequence of the "small" structural ribosomal RNA ("13S") of hamster cell mitochondria, using a procedure involving [3H]isoniazide labeling of samples subjected to sequential periodate oxidation and beta-elimination. The terminus was found to be PyUAUUAOH, which is similar, but not identical, to the corresponding terminus of eukaryotic cytoplasmic 18S rRNA.
Twenty-nine knees with unicondylar, sixty-four with duocondylar, fifty with Guepar, and fifty with geometric prostheses were studied. The follow-up ranged from two to three and one-half years. The unicondylar prosthesis was used in the mildest cases and gave the least complications, but the quality of results was not superior to that achieved with the other prostheses. The duocondylar model was best suited for knees with rheumatoid arthritis and mild deformity. The geometric prosthesis was the best condylar prosthesis for osteoarthritis with moderate to severe deformity, but gave the worst results in knees with rheumatoid arthritis. The Guepar prosthesis was used in the worst knees and gave the best results, but it had the highest infection rate and was the most difficult to salvage. A radiolucency was observed in about 60 per cent of the condylar replacements around the tibial component and in 45 per cent of the Geupar replacements around the femoral component. The significance of this cannot yet be determined but it suggest that the fixation may not be ideal. In all types, residual pain was most frequently attributed to the patellar compartment. Patellectomy was not a solution.
This is a report of 94 knees in 88 patients with the duo-condylar type of knee arthroplasty. The follow-up period of time was between 2 to 4 years with an average of 3 years. The rheumatoid to osteoarthritic patient ratio was 3 to 1. The overall results were excellent in 37.5 per cent, good in 37.5 per cent, fair in 16 per cent, and poor in 9 per cent. The main causes of failure and poor results were: (1) under or over correction of deformity leading to subluxation and/or instability of the knee; (2) loosening of the tibial component, and (3) symptoms arising from the patellofemoral joint. The revision rate is 5.5 per cent. The progressive radiolucency at the cement bone bond is 26 per cent of which 16 per cent is up to 1 mm and 10 per cent is between 1.5 to 3 mm. To further improve the results of arthroplasty, one should take into consideration (1) replacement of the patellofemoral joint, (2) insertion of the prosthesis in the proper anatomical location under correct tension of the ligaments and capsule with the help of proper instrumentation and (3) improvement in fixation of the tibial component.
The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequences obtained were: Bacillus stearothermophilus, -G(Z)approximately 5 Y-U-C-C-U-U-U-C-U (A); B. subtilis, -G(Z)approximately 7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aerugionosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B stearothermophilus and B. subtilis, -Y-C; C. crescentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis.
Explore the source record for details and available documents.
The sequence of the 3'-terminus of 16S RNA from different bacteria has been determined. Complementarity relationships between this sequence and a purine-rich tract in the ribosome binding site of different bacterial mRNAs suggest that the 3'-end of 16S RNA determines the intrinsic capacity of ribosomes to translate a particular cistron.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The 3'-terminal sequence of 18S ribosomal RNA from Drosophila melanogaster and Saccharomyces cerevisiae was determined by stepwise degradation from the 3'-terminus and labelling with [(3)H]isoniazid. The sequence G-A-U-C-A-U-U-A(OH) was found at the 3'-terminus of both 18S rRNA species. Less extensive data for 18S RNA from a number of other eukaryotes are consistent with the same 3'-terminal sequence, and an identical sequence has previously been reported for the 3'-end of rabbit reticulocyte 18S rRNA (Hunt, 1970). These results suggest that the base sequence in this region is strongly conserved and may be identical in all eukaryotes. As the 3'-terminal hexanucleotide is complementary to eukaryotic terminator codons we discuss the possibility that the 3'-end of 18S rRNA may have a direct base-pairing role in the termination of protein synthesis.
The 3'-terminal sequences associated with the large rRNA complex from a range of eukaryotes were determined after pancreatic or T(1)-ribonuclease digestion of RNA terminally labelled with [(3)H]isoniazid. In all higher eukaryotes examined except Drosophila melanogaster, the 3'-terminal sequences Y-G-U(OH) and G-C-U(OH) were demonstrated for the large RNA component(s) and for 6S RNA respectively. The 3'-terminal sequence of Saccharomyces cerevisiae 26S RNA was Y-G-U(OH) and that of 6S RNA Y-A-U-U-U(OH). Three 3'-terminal sequences were found in equimolar amounts in the heat-dissociable 26S rRNA characteristic of insect ribosomes. These were Y-G-U-G-U(OH), Y-C-G-U(OH) and G-C-U(OH) for cultured Antheraea eucalypti cells, Y-G-U(OH), Y-G-U(OH) and G-C-U(OH) for Galleria mellonella larvae and Y-C-G-A(OH), Y-G-U-A(OH) and G-Y-U-G(OH) for Drosophila melanogaster flies. Thus the introduction of the central scission in insect 26S rRNA results in the generation of a unique 3'-terminus and does not arise from random cleavage of the polynucleotide chain.