Segregation of all four major fibrillar collagen genes in the Marfan syndrome.

Linkage markers at or close to the genes encoding the three major fibrillar collagens were used to analyze the segregation of these loci in six pedigrees with dominantly inherited Marfan syndrome. Four pedigrees were discordant at one of the Type I collagen loci (COL1A2), and, of these, two were discordant at the other Type I locus (COL1A1). The Marfan syndrome also segregated independently of the structural loci for Type II and Type III collagen in these two families. This is evidence against the Marfan syndrome being, in general, due to mutations in the major fibrillar collagen genes.     

A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.     

Homozygous osteogenesis imperfecta unlinked to collagen I genes

Summary In a consanguineous pedigree in which a severe type of osteogenesis imperfecta was segregating as an autosomal recessive trait, analysis of genetic markers for both collagen I structural loci COL1A1 and COL1A2 showed that the phenotype was unlinked to either locus.     

Prevention of guanine modification and chain cleavage during the solid phase synthesis of oligonucleotides using phosphoramidite derivatives.

Phosphoramidite reagents can phosphitylate guanine bases at the O6-position during solid phase synthesis and serious chain cleavage occurs if the base phosphitylation is not eliminated before the iodine/water oxidation step. This can be accomplished by blocking the O6-position with a 2-cyanoethyl protecting group for deoxyribonucleotides or with a p-nitrophenylethyl group for ribonucleotides, regenerating the guanine base with water or acetate ions, or using N-methylanilinium trifluoroacetate (TAMA) as the phosphoramidite activator. The effectiveness of these methods was demonstrated by both 31P NMR studies and by the synthesis of d(Gp)23G, (Gp)14G, and d-(Gp)13rG sequences.     

Diadenosine 5′,5‴-P1,P3-triphosphate in eukaryotic cells: Identification and quantitation

A highly sensitive enzymatic assay for diadenosine 5′,5?-P¹,P³-triphosphate (Ap₃A) has been established on the basis of the coupled luminescence assay for diadenosine 5′,5?-P¹,P³-tetraphosphate (A. Ogilvie (1981)Anal. Biochem.115, 302–307). Snake venom phosphodiesterase splits Ap₃A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap₃A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap₃A in various eukaryotic cells after ion-exchange chromatography and high-performance liquid chromatography of acidic extracts of the cells. The level of diadenosine triphosphate was higher in all instances than the level of diadenosine tetraphosphate. When growing in the abdominal cavity of mice, Ehrlich ascites tumor cells contained high amounts of Ap₃A ($\text{0.1}\text{nmol}\text{10}{}^6\text{cells}$), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap₃A with the luminescence assay gave identical results. Ap₃A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap₃A in biological material.     

Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization

Diadenosine tetraphosphatase, an enzyme splitting diadenosine tetraphosphate to AMP and ATP, has been purified to apparent homogeneity from a permanent cell line derived from a leukemic child. The purification procedure consisted of fractionation by ammonium sulfate precipitation, followed by Sephacryl 200 and DEAE-cellulose chromatography, and finally a differential membrane filtration. The enzyme is a single polypeptide chain of Mr = 17,500 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weight of the native enzyme was calculated as 20,000 from gel filtration data. The apparent Km for Ap4A was 0.5 microM as determined by two independent kinetic assays. None of the following compounds were substrates of the enzyme: diadenosine triphosphate, NAD, nucleoside 5'-phosphates (AMP, ATP, GDP, GTP, and UTP). The enzyme had optimal activity in the presence of 1 mM Mg2+, showing no activity in the presence of EDTA.