Robert C. Shuster

Mitochondrial DNA in anucleate human blood cells

Homogeneous populations of human blood platelets or erythrocytes were lysed in alkaline EDTA, bound to nitrocellulose and hybridized to a radioactive mtDNA probe. By comparison to standards of known mtDNA concentration, we determined that platelets contained 4 mtDNA molecules per cell. Rhodamine 123 staining revealed an average of 4 mitochondria per platelet indicating that each mitochondrion contains a single mtDNA molecule. No detectable mtDNA was found in erythrocyte lysates. Using the same procedure, we found that in nucleated cells, mitochondria contained multiple mtDNAs per mitochondrion.

Increased glucose transport by human fibroblasts with a heritable defect in insulin binding.

Insulin and IGF-I binding and their regulation of hexose transport were evaluated in skin fibroblasts cultured from a family (Atl) whose proband had leprechaunism, hypoglycemia, and severe insulin resistance. High affinity insulin binding to proband Atl cells was absent, and partially, but equally, impaired in fibroblasts from his related parents. IGF-I binding to his cultured fibroblasts was within the normal range. Cells from proband Atl had insulin receptor mRNAs similar to control fibroblasts. 3-O-Methyl-D-glucose (OMG) transport by proband Atl was threefold higher than in control fibroblasts (37.7 v 7.6-11 nmol/mL/s) and was insulin-insensitive. Proband Atl fibroblasts had a threefold increase in the Vmax for OMG entry and a concomitant increase in the number of D-glucose-inhibitable cytochalasin B binding sites on their plasma membrane. Similar levels of glucose transporter mRNA were observed in control and proband Atl fibroblasts. These results suggest that fibroblasts from patient Atl have a genetically transmitted mutation in the alpha subunit of their insulin receptor. In the homozygous affected proband, this mutation impairs insulin binding and causes elevated, insulin-insensitive glucose transport. The dysfunction resulting from this mutation is similar to that introduced in Chinese hamster ovary cells by transfection with a truncated alpha subunit.

Insulin-receptor autophosphorylation and kinase activity are constitutively increased in fibroblasts cultured from a patient with heritable insulin-resistance

Mutations in the insulin receptor gene have been described in families with the inherited insulin-resistant syndrome leprechaunism. At a cellular level, these mutations result in decreased insulin binding and impaired insulin stimulation of receptor autophosphorylation and sugar transport. By contrast, we previously found that fibroblasts cultured from leprechaun patient Atl had constitutively increased sugar transport, even though insulin binding was markedly reduced. Here we report that these fibroblasts have basal insulin-receptor autophosphorylation and kinase activity constitutively increased above insulin-stimulated control cells.

The effect of chloramphenicol and rifampicin on the “nicking” and “membrane attachment” of bacteriophage λ DNA☆

Abstract The cleavage of the closed circular form of λ phage DNA in infected cells is dependent upon functions specified by phage genes O and P . The addition of either chloramphenicol or rifampicin to a culture of induced lysogenic bacteria rapidly inhibited the destruction of closed circular DNA from a superinfecting phage indicating that the endonucleolytic activity controlled by genes O and P is unstable. However, the derepressed prophage was able to complement the nicking activity of a superinfecting O − or P − phage for up to 1 hr after induction, suggesting that the O and P gene products are continually synthesized even though they are metabolically labile. Induced lysogenic cells were superinfected with λc 17 which contains a new promotor site and is constitutive for rightward transcription. The superinfecting λc 17 DNA formed a rapidly sedimenting “membrane complex” in the presence of chloramphenicol even though nicking of closed circular DNA was inhibited. The inhibition of nicking after chloramphenicol addition is, therefore, not due to limited amounts of an unstable gene N protein. In contrast to chloramphenicol, rifampicin inhibited the membrane binding of super-infecting λc 17 DNA suggesting that transcription of the phage DNA enhances membrane complex formation.

DNA synthesis in cells infected with phage P1c3

Abstract Temperature sensitive P1 c 3 mutants do not synthesize normal amounts of DNA at the nonpermissive temperature.

On the role of the dnaB protein of Escherichia coli in the replication of ? bacteriophage DNA

SummaryThe interaction between the dnaB protein of E. coli and the gene P product of λ bacteriophage was investigated by measuring the cleavage of closed circular phage DNA after infection of two temperature sensitive dnaB mutants, JG28 and To534 groP-B. Cleavage of superhelical DNA from a λπB mutant phage was observed after infection of either strain whereas superhelical DNA from a wild type phage was only cleaved after infection of JG28. When DNA synthesis in infected cells was blocked by incubation at the nonpermissive temperature, no inhibition of superhelical phage DNA cleavage was observed. It is concluded that in conditions where the dnaB protein has lost the capacity to function in nucleotide polymerization, it is capable of interacting with the λ replication gene products to introduce a break in the phage DNA.

Evidence that the cro repressor inhibits expression of the bacteriophage ?P gene at high multiplicities of infection

SummaryThe activity of the λP gene product at various multiplicities of infection (m.o.i.) was examined in C1-conditions using an assay which measures the disappearance of the rapidly-sedimenting closed-circular (c.c) form of phage DNA. When cells were infected with λC1857 at multiplicities of 5 phage/cell or less, between 65%–75% c.c. DNA was lost during incubation. If the multiplicity of infection was increased to 10 phage/cell or greater, a marked inhibition in the cleavage of c.c. DNA was observed.When bacteria were infected with either λCI857CII2002 or λCI857cro27 at low m.o.i., the usual 65%–75% decrease in the percentage of c.c. phage DNA occurred during incubation. In contrast, no losses in c.c. DNA were noted after infection with λCI857cro27susP3. At high m.o.i., the cleavage of c.c. DNA was inhibited after infection with λCI857CII2002, but not after infection with λCI857cro27. It is concluded that at high m.o.i. in C1-infections, the expression of gene P is unaffected by the CII gene product, but is inhibited by the increased intracellular levels of cro protein.