Oligodeoxynucleotide-directed cleavage and repair of a single-stranded vector: a method of site-specific mutagenesis

A simple and efficient site-specific mutagenesis method is described. First, a single-stranded (ss) circular vector is linearized at the site where the desired mutation will be introduced. To do this, an oligodeoxynucleotide complementary to the target region of the ss vector and containing a restriction enzyme recognition sequence is annealed to the circular ss vector, and the partial double-strand formed is subsequently cleaved with that enzyme. Then, another oligodeoxynucleotide spanning the nick and carrying the mutation is annealed to the linearized ss DNA template and the annealed mixture is used directly to transform Escherichia coli without prior enzymatic DNA synthesis in vitro. The procedure has been applied successfully to constructing insertion, deletion, and point mutations in both M13 phage vectors and plasmid vectors containing the f1 origin of replication.     

Distribution of lipid-binding regions in human apolipoprotein B-100

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.     

抗阿特拉津转基因大豆植株后代的遗传分析

本试验用阿特拉津溶液涂抹、荧光诱导动力学检测、分子杂交等方法对抗阿特拉津转基因大豆植株的后代进行了鉴定,在第二代及第三代中检测到了抗性基因的存在,表明从龙葵中得到的此抗阿特拉津 psbA 基因不仅能导人大豆叶绿体基因组中获得表达,而且可以遗传到后代。     

Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.     

The macrosatellites of the Toulouse goose: the major tandemly repetitive DNA in the toulouse goose genome

This paper examines the principal classes of repetitive DNA of the Toulouse goose (Anser anser) genome. There are four major classes and they are tandem repeats of less than 200 base pairs (bp). The longest repeat (class A) is 190 bp long and starts with a HinfI site. Class B is 43 bp long, commencing with a FokI site. Classes A and B show no extensive homology to DNA sequences held on a current data base (Genbank) but were confirmed to exist as major repeats in another strain of goose, the Emden goose (Anser anser) genome. Classes C and D are 5-bp repeats of 5' GAGAG 3' and 5' GGGAA 3', respectively. The macrosatellites C and D were compared with a current data base (Genbank) and were found to exist in a variety of other organisms as satellites.     

Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae.

Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III. Only one origin was detected, and that origin is located within several hundred bp of an ARS element.     

The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence

We have purified a protein (TEF-1) that specifically binds to two sequence unrelated motifs (GT-IIC and Sph) of the simian virus 40 (SV40) enhancer. TEF-1 binds cooperatively to templates containing tandem but not inverted or spaced repeats of its cognate motifs. This cooperative binding correlates with the ability of the tandem repeats to generate enhancer activity in vivo. In contrast, TEF-1 and a second SV40 enhancer binding protein, TEF-2, bind independently to templates containing the cognate motifs of both proteins (GT-I and either GT-IIC or Sph motifs) even though these motifs cooperate in enhancer activity in vivo. These results allow us to distinguish different classes of enhancer factors.     

On the generalization of the Weinbaum-Jiji bioheat equation to microvessels of unequal size; the relation between the near field and local average tissue temperatures

The extensive series of experiments reported in Lemons et al. [1] show that measureable local tissue temperature fluctuations are observed primarily in the vicinity of the 100-500 micron countercurrent vessels of the microcirculation and thus strongly support the basic hypothesis in the new bioheat equation of Weinbaum and Jiji [2] that these countercurrent microvessels are the principal determinants of local blood-tissue heat transfer. However, the detailed temperature profiles in the vicinity of these vessels indicate that large asymmetries in the local temperature field can result from the significant differences in size between the countercurrent artery and vein. Using the superposition techniques of Baish et al. [9], the paper first presents a solution to the classic problem of an unequal countercurrent heat exchanger with heat loss to the far field. This solution is then used to generalize the Weinbaum-Jiji bioheat equation and the conductivity tensor that appears in this equation to vessels of unequal size. An asymptotic analysis has also been developed to elucidate the relationship between the near field temperature of the artery-vein pair and the local average tissue temperature. This analysis is used to rigorously prove the closure approximation relating the local arterial-venous temperature difference and the mean tissue temperature gradient which had been derived in [2] using a more heuristic approach.     

Effects of traces of rare earth elements on the protozoanBlepherisma and on mice

The growth of the protozoanBlepherisma is stimulated by Lanthanum (La) at concentrations as low as 0.32 ppm. In mice Yttrium (Y) and Ytterbium (Yb) are absorbed, accumulated, and metabolized. Both rare earth elements (RE) exhibit a high affinity for teeth and bones, accumulation occurs and metabolism is slow. In the livers of RE-exposed mice, concentrations are variable. The liver is apparently capable of absorbing and discharging RE in a manner depending on metabolic activity. The main route of discharge for ingested REs is the alimentary canal. Exposure of pregnant mice to RE leads to rapid placental transfer of RE; 14.1% of the total amount of RE administered was detected in newborn mice. Young, developing organisms appear to be especially susceptible to RE accumulation.