Polymerase chain reaction (PCR) techniques for site-directed mutagenesis

Polymerase chain reaction (PCR) technology has revolutionized the process of isolating and amplifying segments of DNA. One powerful application of PCR is its use in precise site-directed mutagenesis (SDM). SDM provides an elegant tool for scientists and engineers to explore biocatalytic mechanisms and processes to understand the structural-functional relationships of enzymes and other proteins. This article reviews techniques and methodology used in site-directed mutagenesis of genes by PCR.     

Importance of communal foraging grounds outside the reed marsh for breeding great reed warblers

Foraging habitat selection of breeding great reed warblers was studied at a shore of Lake Biwa. The foraging grounds of parent warblers during the nesting period were not restricted to the breeding territory of the reed marsh, their nestling habitat. The paddy field outside the reed marsh was used communally by them throughout the breeding season. Females with early stage nestlings did not visit the paddy field whereas when nestlings were older than 3 days, more than half of their total food was collected there. Females with nests adjacent to the paddy field tended to exploit the paddy field more often than those with nests distant from it. Monogamously mated females tended to exploit the paddy field more often than polygynously mated females. Food collected in the paddy field was larger than that in the reed marsh and parent birds were prepared to travel longer distances to exploit the rich source of food in the paddy field. The importance of the communal foraging ground outside the reed marsh as a background of the polygynous mating system of this species is discussed.     

Universal primers for amplification of three non-coding regions of chloroplast DNA

Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.     

Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Inhibited growth of common enteropathogenic bacteria in lactic-fermented cereal gruels

A natural lactic fermentation of mixtures of water and whole flour of either maize or high-tannin sorghum was obtained either before or after cooking to a weaning gruel: The preparations had a final pH of about 3.8 (range 3.67 to 4.00) and a ratio of lactic acid to acetic acid of 91 (w/w). The growth of added (about 10⁷ c.f.u./g gruel) Gram-negative intestinal pathogenic bacteria, enterotoxigenicEscherichia coli, Campylobacter jejuni, Shigella flexneri andSalmonella typhimurium, was strongly inhibited in the sour gruels, and the effect could primarily be explained by the low pH caused by the formation of lactic and acetic acids during the fermentation process. Of the added Gram-positive bacteria,Bacillus cereus andStaphylococcus aureus showed similar inhibited growth up to 7h after inoculation in the sour gruels. The strain ofStaphylococcus, however, showed only a continued reduction in growth in the fermented gruel samples, which had a viable lactic bacteria culture indicating the presence of a bacteriocin. This implies that a low pH (< 4.0) alone is not sufficient to sustain the inhibition of the growth ofStaphylococcus aureus. The survival studies were carried out at optimal temperatures for each respective enteropathogen.     

渤海南部半滑舌鳎的食性及摄食的季节性变化

本文研究了渤海南部半滑鳎的食性及其摄食的季节性变化,胃食物分析结果表明,半滑舌鳎为底栖生物食性鱼类,以虾类,蟹类,双壳类及部分中下层小型鱼类为主要食物,兼食一些多毛类,头足类,腹足类,棘皮动物及海葵,半滑舌鳎终年摄食,其摄食强度的周年变化不大,半滑舌鳎常年捕食鲜明鼓虾,日本鼓虾,口虾蛄,隆线强蟹及泥古隆背蟹等。夏季加强捕食小刀蛏,凸壳肌蛤等双壳类,降春季外还捕食一些以虾虎鱼为主的中下层小型鱼类。     

人α1/158Ⅴ型(α1c型)干扰素基因在大肠杆菌中的表达及其产物的初步纯化

采用DNA聚合酶链反应(PCR)技术,将本实验室从中国人胎肝细胞染色体DNA中发现和分离的IFN-α1/158V基因的原始克隆,改造成适于进行非融合蛋白原核表达的结构形式,并在大肠杆菌中获得高效表达。测得重组IFN-α1/158V的抗病毒活性为1.9×10~7单位/升菌液。随后又采用以单克隆抗体亲和层析为主的纯化流程对表达产物进行初步纯化,获得了在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带的纯化产物。     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

Phagotrophic phytoflagellates in microbial food webs

Phagotrophy by pigmented flagellates is known from the literature but has recently been rediscovered in the context of microbial food webs. Particle ingestion rates were found to be equivalent for pigmented and nonpigmented microflagellates in both field and laboratory studies. Ingestion rates of the chrysophytes Ochromonas danica, O. minuta, and Poterioochromonas malhamensis, the dinoflagellate Peridinium inconspicuum, and the cryptophytes Cryptomonas ovata and C. erosa were compared with those of two nonpigmented Monas species using 0.57 μm polystyrene beads as a food source. Ingestion rates were 0.31 to 3.17 beads/cell/h and filtration rates were 10⁻⁷ to 10⁻⁸ ml/cell/h with no detectable difference between pigmented and nonpigmented forms. Ingestion rates in unpigmented Monas species showed a linear increase with increasing particle concentration from 1.9 × 10⁶ to 1.6 × 10⁷ beads/ml. Light and DOC levels in the range of those encountered by phytoflagellates in the field also influenced laboratory measurements of bead ingestion by Poterioochromonas malhamensis. Ingestion rates decreased and photosynthesis increased over the natural PAR light range from 0 to 1800 microeinsteins/s/m². At 40 microeinsteins/s/m² maximum ingestion rates and high rates of photosynthesis occurred simultaneously. Ingestion rates decreased above 4 mgC/l supplied as glucose. DOC levels commonly occurring in Lake Oglethorpe range from 3.5 to 10.0 mgC/l. These studies suggest that mixotrophy, the trophic utilization of particulate food and dissolved organic matter as well as photosynthetically fixed organic matter, is a balanced process that can be regulated by environmental conditions. In field studies during a chrysophyte bloom, phytoflagellate grazing exceeded heterotrophic microflagellate grazing and constituted up to 55% of the bactivory of all microflagellates, ciliates, rotifers, and crustaceans combined. Neither bacterial abundance, light nor temperature were good predicters of grazing rates for the phagotrophic phytoflagellate association as a whole during this unstratified period. Phagotrophs are often most abundant at the metalimnetic plate during stratification.