The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing.

Transposon gamma delta (Tn1000), a 6-kb member of the Tn3 family, is widely used for plasmid mutagenesis. A 1.8-kb derivative of gamma delta was constructed that contains the kan gene from Tn5 and the resolution (res) site from gamma delta cloned between 40-bp inverted repeats of gamma delta's delta (delta) end. This element, named m gamma delta-1, lacks the genes encoding transposase and resolvase, and therefore depends on its host to supply transposition and resolution functions. Thus, in strains lacking gamma delta, m gamma delta-1 will not transpose. The m gamma delta-1 element is shown to be useful for mutagenesis of plasmids, DNA sequencing, and allele replacement (in Streptomyces avermitilis).     

Use of helper-free retroviral vector to direct a high expression of porcine growth hormone in mouse fibroblast cells.

A retroviral vector has been employed to express the cDNA coding for porcine growth hormone (pGH) in the mouse fibroblast cell NIH 3T3 in large quantity. In this study, a single gene vector which contained no selectable marker was used. We have coinfected NIH 3T3 cells with pGH retrovirus and Neo(r) retrovirus to obtain a stable, high-expression clone. Using a superinfection strategy, we further increased the copy number of proviral DNA in the host chromosome, thus increasing the pGH secretion from 22 to 55 micrograms/10(6) cells/24 h. The recombinant pGH produced from mouse fibroblast cells was heterogeneous at the N-terminus, which mimicked the situation with bovine growth hormone either from natural sources or from recombinant products derived from mouse fibroblasts. This technology is useful for many biologically important genes to be stably transduced by retroviral vector into mammalian cells and highly expressed.     

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.     

Protection by salvianolic acid A against adriamycin toxicity on rat heart mitochondria.

It was found that salvianolic acid A (Sai A) has potent antioxidant activity. The effects of Sai A on adriamycin-induced heart mitochondrial toxicity of rats in vitro and on adriamycin antitumor activity are investigated in this article. Malondialdehyde (MDA) formation and membrane rigidification of rat heart mitochondria intoxicated with adriamycin were significantly reduced by Sai A. In the electron spin resonance (ESR) studies, Sai A has no significant effect on the formation of adriamycin semiquinone radicals (AQ.), while hydroxyl radicals generated by electron transfer from AQ. to H2O2 were scavenged by Sai A dose-dependently. On the other hand, Sai A was shown to have no effects on the antitumor activity of adriamycin in cultured L1210 ascitic tumor cells and in mice with P388 ascite tumor. These results indicate that Sai A protects against adriamycin induced heart mitochondrial toxicity of rats, while Sai A has no antagonizing effect on the antitumor activity of adriamycin.     

Direct detection of circulating free radicals in the rat using electron spin resonance spectrometry.

We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.     

Analysis of point mutations induced by ultraviolet light in human cells

Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Five large independent cultures of human lymphoblastoid cells, line TK6, were exposed to 4 J/m2 of 254-nm UV light and mutants at the HPRT locus were selected en masse by 6-thioguanine (6TG) resistance. Exon 3 of the HPRT gene was amplified from the mutant cells by polymerase chain reaction (PCR) using modified T7 DNA polymerase. Denaturing gradient gel electrophoresis (DGGE) was used to separate the mutant sequences from the wild type as mutant/wild-type heteroduplexes. Individual mutant bands were isolated from the gel and the nature of the mutations was determined by direct sequencing. Eight predominant mutations were detected in the 184-bp exon 3 sequence. Of these, 3 transition, including 2 G-C to A-T and 1 A-T to G-C and 2 A-T to C-G transversions, appeared in all 5 UV-treated cultures but not in untreated cultures and were thus considered to be mutational hotspots. These observations are similar in nature to those previously reported in bacterial and rodent cells. A single G deletion, a tandem substitution of CpT for TpA, and a tandem triple substitution of GpGpA for ApApG were also observed but in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Numerical analysis of the mutant fractions of these 8 mutations indicated that each of them was distributed as a set of non-random and independent events, i.e., a mutational hotspot.     

Technique for repeated collection of cerebrospinal fluid from cisterna magna of anesthetized strain 13 guinea pigs

To study biochemical changes in cerebrospinal fluid (CSF), we developed a reliable technique for repeated collection of CSF in anesthetized strain 13 guinea pigs. The animal's head was mounted in a stereotaxic instrument with ventral tilt at 30 degrees, and cisternal puncture was made with an L-shaped, 23-gauge needle through the shaved skin. Clear CSF was collected in a 1-ml syringe surrounded by crushed ice. Each collection procedure lasted for 3 min, and three consecutive collections produced about 0.2 ml of CSF. Sampling was repeated at 3-hr intervals. With intravenous saline infusion (10 ml/kg.hr), a total volume of 0.6-1.0 ml of CSF was collected over 6 to 12 hr. Animals maintained a mean blood pressure, heart rate, and minute volume, with few changes during CSF sampling for the entire collection.