739. Genomic DNA Reduction for Therapeutic DNA Manufacture

Sulfate is a primary source of sulfur for most microbes and in some prokaryotes it is used an electron acceptor. The acidophileFerroplasma acidarmanus(strain fer1) requires a minimum of 150 mM of a sulfate-containing salt for growth. Sulfate is assimilated byF. acidarmanusinto proteins and reduced to form the volatile organic sulfur compounds methanethiol and dimethyldisulfide. In the absence of sulfate, cell death occurs by an unknown mechanism. In this study, cell viability and genomic DNA and ATP contents ofF. acidarmanuswere monitored in response to the absence of sulfate or the presence of sulfate and the sulfate analog molybdate (MoO42-). Cellular DNA and ATP contents were monitored as markers of cell viability. The absence of sulfate led to a decrease in viable cell numbers of greater than 7 log10within 5 days, a > 99% reduction in genomic DNA within 3 days, and a > 60% decrease in ATP within 6 h. Likewise, cells incubated with lost viability (decreased by > 2 log10in 5 days), extractable genomic DNA (reduction of > 60% in 2 days), and ATP (reduction of > 70 % in 2 hours). These results demonstrate that sulfate deprivation or the presence of molybdate have similar impacts on cell viability and essential biomolecules. Sulfate was coupled to cellular ATP content and maintenance of DNA integrity inF. acidarmanus, a finding that may be applicable to other acidophiles that are typically found in sulfate-rich biotopes.

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Paris I Dysfibrinogenemia: A Point Mutation in Intron 8 Results in Insertion of a 15 Amino Acid Sequence in the Fibrinogen γ-Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651583 ◽

1993 ◽

Vol 69 (03) ◽

pp. 217-220 ◽

Cited By ~ 12

Author(s):

Jonathan B Rosenberg ◽

Peter J Newman ◽

Michael W Mosesson ◽

Marie-Claude Guillin ◽

David L Amrani

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Point Mutation ◽

Genomic Dna ◽

Comparative Sequence Analysis ◽

Chain Gene ◽

Molecular Defect ◽

Nucleotide Position ◽

Normal Individuals ◽

Polymerase Chain

SummaryParis I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal γ-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the γ-chain region of the Paris I subject’s genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A→G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I γ-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature γparis I chain mRNA, and encodes a 15 amino acid insert after γ350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I γ-chain mRNA also results after translation into a substitution of S for G at position γ351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the γpariS I chain. We conclude that the insertion of this amino acid sequence leads to a conformationallyaltered, and dysfunctional γ-chain in Paris I fibrinogen.

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