Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

A method for flow cytometric cell cycle analysis of normal and psoriatic human epidermis based on a detergent/citric acid technique for suspension of nuclei

A new method is described for flow cytometric cell cycle analysis of normal and psoriatic human epidermis, based on non-enzymatic tissue disaggregation. The epidermis was isolated by treatment with acetic acid and stored by freezing. After thawing, the epidermis was disintegrated into a nuclear suspension by 3 steps: incubation with dithiotreitol, whirling in a buffer (pH 7.4) with the non-ionic detergent Nonidet P40, EGTA, RNase and spermine, and whirling after addition of citric acid to a final concentration of 1% (pH 2.4). The suspension was stained with propidium iodide and filtered before flow cytometry. The yield of suspended nuclei was approximately 70% of the original number of cells in the tissue. The detergent/citric acid method was found to be preferable to an ultrasonication method previously used on human epidermis. All cell cycle and cell maturation stages were represented in the detergent/citric acid suspension, in contrast to the selection of immature G1, S and G2 stages with enzymatic methods. In the analysis of psoriatic epidermis inadequately matured (parakeratotic) cells were present in the suspension and had to be discriminated by gating on light scattering intensity, as they were not susceptible to lysis and did not stain properly. The fraction of S phase nuclei was on average 1.9% in normal and 7.7% in psoriatic epidermis, thus confirming the results of other investigators using enzymes. The presence of mitotic figures in the suspension was demonstrated by flow sorting. In this way the mitotic fraction was estimated to 0.06% in normal and 0.22% in psoriatic epidermis, confirming histological data of other investigators.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Background  

Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS) is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference.     

The posthatch physiology of the turkey poult--II. Hematology

Packed cell volume, total leukocytes, total erythrocytes, hemoglobin, mean cell volume and mean corpuscular hemoglobin concentration of the poult were analyzed daily for the first 10 days posthatch. Sexually dimorphic effects on hematological parameters were not apparent, but there was a sex X day interaction for all parameters except hemoglobin indicating that males were slower to develop/respond to critical stimuli. The study revealed a latency in production of erythrocytes and leukocytes in posthatch males and females which coincided temporally with early poult mortality.     

The fifteenth- and twentieth-century colonization of the Basin of Mexico by the Great-tailed Grackle (Quiscalus mexicanus)

Historical evidence indicates that Great‐tailed Grackles colonized the Basin of Mexico from the Gulf Coast lowlands in the fifteenth century. They were probably assisted by an intentional introduction, but colonization succeeded because of anthropogenic habitat alterations over the previous two centuries. During the Colonial period, grackles withdrew from the Basin, only to recolonize it in recent decades. This withdrawal was also due probably to changes in land use, including drainage of much of the water from the Basin's lakes.     

Fine‐scale Population Genetic Structure of Two Dioecious Indian Keystone Species,Ficus hispida and Ficus exasperata (Moraceae)

Although Ficus (Moraceae) is a keystone plant genus in the tropics, providing resources to many frugivorous vertebrates, its population genetic structure, which is an important determinant of its long‐term survival, has rarely been investigated. We examined the population genetic structure of two dioecious fig species (Ficus hispida and Ficus exasperata) in the Indian Western Ghats using co‐dominant nuclear microsatellite markers. We found high levels of microsatellite genetic diversity in both species. The regression slopes between genetic relationship coefficients (f_ij) and spatial distances were significantly negative in both species indicating that, on average, individuals in close spatial proximity were more likely to be related than individuals further apart. Mean parent–offspring distance (σ) calculated using these slopes was about 200 m in both species. This should be contrasted with the very long pollen dispersal distances documented for monoecious Ficus species. Nevertheless, overall population genetic diversity remained large suggesting immigrant gene flow. Further studies will be required to analyze broader scale patterns.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.