Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham     

Functional regulation of brain pyruvate dehydrogenase: Postnatal development, anesthesia and food-deprivation

The activity of brain pyruvate dehydrogenase complex (PDHC), is regulated by reversible phosphorylation of the alpha subunit of the E1 component (pyruvate dehydrogenase, EC 1.2.4.1) of PDHC. Using an in vitro back-titration assay, we have evaluated the postnatal development of E1 phosphorylation, as well as the effects of acute pentobarbital administration and food-deprivation on cerebral cortical E1 phosphorylation in synaptosomal and free mitochondrial compartments of the albino rat. Between birth and postnatal day 25, the back-titration phosphorylation increased ca 4-fold, with the largest increase occurring between days 15 and 20. The phosphorylation of E1 in the synaptosomal, but not free mitochondrial fraction, was decreased during pentobarbital anesthesia. Following 72 h of food-deprivation, E1 phosphorylation was decreased in both subcellular fractions.

The postnatal increase in E1 back-titration phosphorylation is consistent with and similar in magnitude to previously reported increases in the specific enzymatic activity of PDHC. These results also highlight the potential importance of localized subcellular alterations in mitochondrial metabolism and further validate the back-titration phosphorylation of E1 as a valuable tool for the study of central nervous system PDHC metabolism.     

Assumption validity in human optimal foraging: The Barí hunters of Venezuela as a test case

Optimal foraging theory is being used increasingly as a means of understanding human foraging behavior. One of the central assumptions of optimal foraging theory is that prey items or patches are encountered sequentially and as a Poisson process. Using empirical data gathered on the Barí hunters of Venezuela, we assess the validity of this central assumption. We compare our observed distribution of encounter frequencies with an expected Poisson distribution, utilizing chisquare tests and graphic representations. The results are strikingly consonant with the expected Poisson distribution and lend support to the applicability of optimal foraging models to human hunting behavior.     

Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing.

Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase.     

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.     

An additional editing site is present in apolipoprotein B mRNA.

Human intestinal apolipoprotein (apo) B mRNA undergoes a C to U RNA editing at nucleotide 6666 to generate a translation stop at codon 2153, which defines the carboxy-terminal of apo B48. Here we show that two of eleven human intestinal cDNAs spanning residue 6666 were edited from a genomically-encoded C to a T at residue 6802 as well as at residue 6666. This additional editing converts Thr (ACA) codon 2198 to Ile (AUA). Synthetic RNA including the nucleotide 6802 was edited in vitro by intestinal extracts at 10-15% of the editing efficiency of nucleotide 6666. A sequence is identified as important for recognition by the editing activity. No secondary structural homology was identified between the two edited sites. No other sequence in the region between 6411 and 6893 nucleotides of apo B mRNA was found to be edited in vivo or in vitro. Apo B RNA editing extracts from intestine did not edit maize cytochrome oxidase II mRNA.     

Genetic counselor training: a review and considerations for the future.

The first training program for genetic counselors began in 1969. Since then a number of other programs have been developed and more than 650 individuals have graduated from these programs. This article reviews the development and current status of training opportunities for genetic counselors. Twelve programs that currently grant a master's-level degree in genetic counseling are reviewed. Other areas, such as certification and licensure, that reflect genetic counseling training or such issues of professional growth as continuing education and career advances are addressed.     

DNA polymorphisms of the apolipoprotein AII and AI-CIII-AIV genes: a study in men selected for differences in high-density-lipoprotein cholesterol concentration.

We have investigated the frequencies of RFLPs of the apolipoprotein (apo) AII gene and of the apo AI-CIII-AIV gene cluster in 109 men, selected from a random sample of 1,910 men aged 45-59 years, to cover a wide range of plasma high-density-lipoprotein (HDL)-cholesterol concentration. There was no significant difference in apo AI or apo AII RFLP allele frequency between groups of individuals with high and low HDL-cholesterol concentration. However, the apo AI PstI RFLP showed an association with genetic variation determining the plasma concentration of apo AI in this sample. Genetic variation in the apo AI-CIII-AIV gene region, as defined by haplotypes, accounted for 16% of the phenotypic variance in the apo AI concentration and for 8% of the phenotypic variance in HDL-cholesterol concentration. There was no significant association between alleles of the apo AII MspI RFLP and genetic variation determining apo AII or HDL concentration. The data demonstrate that genetic variation in the apo AI-CIII-AIV gene cluster is involved in determining the serum concentration of apo AI in this sample of clinically well individuals.