End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells.

We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.     

Purification and properties of mycodextranase from Streptomyces sp. J-13-3.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.     

Purification and characterization of an antibiotic substance produced from Rhizopus oligosporus IFO 8631.

We obtained a purified antibiotic protein from the submerged cultivation broth of Rhizopus oligosporus IFO 8631 by using CM-Cellulofine chromatography and HPLC. The antibiotic did not show a broad spectrum of activity, but it was very active against some of the Bacillus species, especially against Bacillus subtillis (B. natto) at a very low concentration (less than 1 ppm). It also showed activity against other gram-positive bacteria, including Staphylococcus aureus and Streptococcus cremoris. The purified antibiotic was a simple protein of about 5,500 in molecular weight, the amino acid component being characteristically high in cystine content. This high cystine content contributed to the stability of the antibiotic over a wide pH range and against strong heating (50% of the activity remained after boiling for 1 hr).     

HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Peripheral resistance and red cell LiNa countertransport in borderline hypertensives

We have studied ouabain-resistant, external sodium-stimulated, lithium efflux (LiNa countertransport) in red blood cells from 21 borderline hypertensives with at least one hypertensive first degree relative (BH-F), 19 borderline hypertensives without family history of essential hypertension (BH-NF), and 35 age-matched normotensive subjects. The data indicate the finding of an increased LiNa countertransport in all BH (F+NF), but with a significant overlap between BH values and control ones: LiNa countertransport is significantly higher only in BH-F but it is normal in BH-NF. Moreover, there is a significant correlation of LiNa countertransport to total peripheral resistance but not to mean blood pressure in all hypertensive patients. It is suggested that in BH the increase of erythrocyte Na flux is mediated by the NaNa exchange diffusion, and its abnormality may be associated to the hereditary trait of essential hypertension rather than the high blood pressure per se, probably resulting in the development of hypertension, through the increased vascular smooth muscle tone.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.