Biomedical subjects
Publications and source records attributed to L Clarke.
Immediate inoculation of HEp-2 cells is generally advocated for the isolation of respiratory syncytial virus. However, delayed laboratory inoculation of properly transported specimens obtained by aspiration of nasopharyngeal mucus provided an isolation rate similar to that obtained with immediate inoculation.
Structure-function analysis of a yeast (Saccharomyces cerevisiae) centromere (CEN3) has been carried out by altering the nucleotide sequence of the DNA within and surrounding the centromere of yeast chromosome III, and observing the behaviour of the resulting altered chromosomes during mitotic and meiotic cell divisions. A centromere substitution vector (pJC3-13) was constructed, which contains in the proper orientation: the DNA sequences that normally flank the chromosome III centromere, a wild-type URA3 gene for selection, and a unique BamHI restriction site for insertion of various DNA sequences to be assayed for centromere activity. Cleavage of the plasmid DNA with EcoRI generates a linear DNA fragment whose ends are homologous with the regions flanking the centromere. Transformation of the appropriate homozygous ura3 diploid yeast strain with this linear DNA results in URA3+ transformants in which the CEN3 region on one copy of chromosome III has been replaced by the URA3 gene and the DNA sequence previously inserted into the vector. These studies identify a 289 base-pair (bp) DNA fragment from the CEN3 region that retains full centromere function when used to replace the normal CEN3 sequence. Centromeres function equally well in either orientation, and the chromosome XI centromere (CEN11) can be used to replace CEN3, with no observable effect on mitotic or meiotic chromosome segregation. Various DNA restriction fragments occurring within the CEN3 region were used alone or in combinations to replace the normal CEN3 sequence. Yeast centromeres contain a high A + T region about 82-89 bp in length (element II) flanked by a highly conserved 11 bp sequence (III) and a less-conserved 14 bp sequence (I). The experiments demonstrate that both regions II and III are necessary for normal centromere function, although centromeres containing III plus truncated or rearranged portions of the high A + T region II retain partial activity. Chromosomes of the latter type often give abnormal segregation patterns through meiosis, including separation and random segregation of sister chromatids during the first meiotic division.
Adsorption of autologous plasma over nonviable Staphylococcus aureus Cowan I (SAC) followed by reinfusion of the plasma causes regression of (a) chemically induced rat mammary adenocarcinomas (MA), and (b) canine transmissible venereal tumors (TVT) and spontaneously occurring dog tumors. Animal data are more impressive than that from trials in humans. Nine of 41 patients receiving perfusions of adsorbed plasma showed some partial objective response. Eight of these nine patients received multiple perfusions over a period of time. Twenty-one of 41 patients showed subjective responses. Adsorption of autologous plasma over non-protein A--containing S. aureus Wood 46 caused regression of MA. Adsorption of normal rat plasma with SAC, and infusion of this adsorbed plasma into mammary tumor--bearing rats, caused regression of MA. Intravenous infusion of purified protein A alone caused regression of both rat MA and canine TVT. Superimposed on this observation is the finding that, during plasma adsorption, bacterial moieties leach from SAC. It is possible that the immunostimulation observed in plasma-perfused hosts is due to the removal of plasma blocking agents on one hand, and introduction of the bacterial agents on the other.
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We determined the nucleotide sequence of DNA segments containing functional centromeres (CEN3 and CEN11) isolated from yeast chromosomes III and XI. The two centromere regions differ in primary nucleotide sequence, but contain structural features in common. Both centromere regions contain an extremely A + T-rich core segment 87-88 bp in length, flanked by two short sequences (14 bp and 11 bp) that are identical in both DNAs. These elements plus one additional 10 bp region of perfect homology are positioned in an almost identical spatial arrangement within the two centromere regions. Significant homologies are also observed among the sequences flanking the high A + T region and various satellite DNA sequences from higher eucaryotes, although no repeated sequences occur near the yeast centromeres. Centromere activity in vivo is maintained on relatively small DNA fragments (627 bp for CEN3 and 858 bp for CEN11), as assayed by mitotic stabilization of autonomously replicating ars plasmids in yeast.
Simultaneous renal clearances of inulin (CIN), p-aminohippurate (CPAH), and creatinine (CCR) were measured in hydrated mares (6 ponies and 2 horses). The CIN and CPAH were determined during steady-state infusion at 3 different infusion rates. A 6-fold change in plasma IN concentration did not produce alteration in CIN, nor was there a difference between the ponies and horses (P greater than 0.2). The overall average (mean +/- SEM) was 190.6 +/- 5.89 ml . min-1 . 100 kg of body weight-1. There was no difference noted between simultaneous CIN and CPAH. Clearance of PAH remained essentially constant during the change in plasma PAH from 0.33 mg/dl to 5.27 mg/dl. The extraction ratio of PAH for the nonanesthetized pony was 0.966. Effective renal plasma flow (CPAH) of the pony exceeded that of the horse.
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An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.
The centromeric DNA (CEN3) from yeast chromosome III has been isolated on a 1.6 kilobase-pair segment of DNA located near the centromere-linked CDC10 locus of Saccharomyces cerevisiae. When present on a plasmid carrying a yeast chromosomal replicator, CEN3 enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing CEN3 are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions.
Urinary oxalate was determined in an ambulatory setting in 107 patients with an increased intestinal calcium absorption rate in whom stones formed, 34 patients with normal calcium absorption in whom stones formed and 34 control subjects without stones. Urinary oxalate excretion was not significantly different when the diet was changed from a random to a calcium-restricted diet. Moreover, urinary oxalate was not higher during summer months when intestinal calcium absorption may have been stimulated. Diet history disclosed that many patients with an increased calcium absorption rate had been on a moderate oxalate-restricted diet, often as part of a calcium-restricted regimen for the control of hypercalciuria. The results indicate that renal oxalate excretion in an ambulatory setting is not critically dependent on the state of calcium absorption and intake, and that the imposition of a low calcium dietary regimen in patients with an increased calcium absorption and in whom stones form does not necessarily augment oxalate excretion.
A hybrid plasmid colony bank was constructed in Escherichia coli using the E. coli-Saccharomyces cerevisiae shuttle vector pLC544 and randomly sheared segments of yeast DNA. By transformation with a hybrid plasmid DNA pool from this collection and complementation of a temperature-sensitive cdc10 mutation in yeast, a plasmid was isolated that carries 8 kilobase pairs of DNA around the chromosome III centromere-linked CDC10 locus. This DNA segment overlaps a larger region of DNA (40 kilobase pairs) previously identified to be around the LEU2 locus on chromosome III [Chinault, A.C. & Carbon, J. (1979) Gene 5, 111-126] and physically establishes the directionality of the cloned DNA sequences with respect to the genetic map and the centromere. In the leu2-cdc10 interval, the relationship between physical distance on the DNA and genetic distance as measured by recombinational frequencies is about 3 kilobase pairs per centimorgan.
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Plasmid pBR313 carrying a 1.4 kb EcoRI fragment from the yeast TRP1 region (designated pLC544) is capable of transforming yeast trp1 mutants to Trp+ at high frequency (10(3)--10(4) transformants/micrograms DNA). Transformation can be achieved either by using purified plasmid DNA or by fusion of yeast spheroplasts with partially lysed Escherichia coli [pLC544] protoplast preparations. The Trp+ yeast transformants are highly unstable, segregating Trp- cells at frequencies of 0.18 per cell per generation (haploids) and 0.056 per cell per generation (diploids) in media containing tryptophan. Plasmid pLC544 replicates autonomously in the nucleus of yeast cells and segregation of Trp-cells is associated with the complete loss of plasmid sequences. In genetic crosses, pLC544 is randomly assorted during meiosis and is carried unchanged through the mating process into haploid recombinants.
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