Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA or tRNA. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA(Pro)-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA(Pro) was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA(Pro) backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.     

Nadine Dobrovolskaïa-Zavadskaïa and the dawn of developmental genetics

In one of the first genetic screens aimed at identifying induced developmental mutants, Nadine Dobrovolskaïa‐Zavadskaïa, working at the Pasteur Laboratory in the 1920s, isolated and characterized a mutation affecting Brachyury, a gene that regulates tail and axial development in the mouse. Dobrovolskaïa‐Zavadskaïa's analysis of Brachyury and other mutations affecting tail development were among the earliest attempts to link gene action with a tissue‐specific developmental process in a vertebrate. Her analyses of genes that interacted with Brachyury led to the discovery of the t‐haplotype chromosome of mouse. After 70 years, Brachyury and the multiple genes with which it interacts continue to occupy a prominent focus in developmental biology research. A goal of this review is to identify the contributions that Dobrovolskaïa‐Zavadskaïa made to our current thinking about Brachyury and how she helped to shape the dawn of the field of developmental genetics. BioEssays 23:365–371, 2001. © 2001 John Wiley & Sons, Inc.     

Effect of exercise intensity on 24-h energy expenditure and nutrient oxidation.

The aim of this study was to determine the effects of exercise at different intensities on 24-h energy expenditure (EE) and substrate oxidation. Sixteen adults (8 men and 8 women) were studied on three occasions [sedentary day (Con), a low-intensity exercise day (LI; 400 kcal at 40% of maximal oxygen consumption) and a high-intensity exercise day (HI; 400 kcal at 70% of maximal oxygen consumption)] by using whole room indirect calorimetry. Both 24-h EE and carbohydrate oxidation were significantly elevated on the exercise days (Con < LI = HI), but 24-h fat oxidation was not different across conditions. Muscle enzymatic profile was not consistently related to 24-h fat or carbohydrate oxidation. With further analysis, it was found that, compared with men, women sustained slightly higher rates of 24-h fat oxidation (mg x kg FFM(-1) x min(-1)) and had a muscle enzymatic profile favoring fat oxidation. It is concluded that exercise intensity has no effect on 24-h EE or nutrient oxidation. Additionally, it appears that women may sustain slightly greater 24-h fat oxidation rates during waking and active periods of the day.     

Bacteriophage strain typing by rapid single molecule analysis

Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.     

Trans-centromere effects on meiotic recombination in the zebrafish

We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.     

Marked effects of salt on estrogen receptor binding to DNA: biologically relevant discrimination between DNA sequences

Avidin-biotin complexed with DNA (ABCD) assays were employed to determine the binding affinity of estrogen receptor (ER) to DNA under various salt conditions. Type and concentration of salt in the reaction buffer dramatically affected the ability of the ER to discriminate between DNA sequences. Under appropriate salt conditions, ER was able to bind to the estrogen response element from the Xenopus vitellogenin A2 gene with at least 3 orders of magnitude greater affinity than a two base pair mutant sequence, and 5 orders of magnitude greater affinity than plasmid DNA. In these studies, the best discrimination was observed under conditions of salt type and concentration that more closely approximated intracellular conditions, i.e., 100-150 mM potassium salts. Analysis of the binding affinities for ER to all three types of DNA over a range of KCl concentrations indicated that the ionic interactions upon ER binding were the same for the three DNA molecules tested. Therefore, the additional stability of ER binding to target DNA sequences was contributed by nonionic interactions.     

Estimates of DNA and protein sequence divergence: an examination of some assumptions

Some of the assumptions underlying estimates of DNA and protein sequence divergence are examined. A solution for the variance of these estimates that allows for different mutation rates and different population sizes in each species and for an arbitrary structure in the initial population is obtained. It is shown that these conditions do not strongly affect estimates of divergence. In general, they cause the variance of divergence to be smaller than a binomial variance. Thus, the binomial variance that is usually assumed for these estimates is safely conservative. It is shown that variability in the mutation rate among sites can have an effect as large as or larger than variability in the mutation rate among bases. Variability in the mutation rate among bases and among sites causes the number of substitutions between two sequences to be underestimated. Protein and DNA sequences from several species are collected to estimate the variability in mutation rates among sites. When many homologous sequences are known, standard methods to estimate this variability can be used. The estimates of this variability show that this factor is important when considering the spectrum of spontaneous mutations and is strongly reflected in the divergence of sequences. Smaller variability is found for the third position of codons than for the first and second codon positions. This may be because of less selective constraints on this position or because the third position has been saturated with mutations for the sequences examined.