Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

白色念珠菌ALS家族基因的克隆与功能分析

利用ＣＸ２ ０．５ｋｂ探针,研究在不同白色念珠菌菌株中ＣＸ２串联重复序列的分布,发现多种白色念珠菌中都含有这个重复序列。为证明ＣＸ２的表达与形态转变有关,选用了多种诱导和非诱导菌丝生长的条件培养白色念珠菌ＳＣ５３１４,然后用串联重复序列作控针进行Ｎｏｒｔｈｅｒｎ杂交分析,证明ＣＸ２ ｃＤＮＡ片段的表达与菌丝形态紧密相关。用它作为探针进行染色体定位和基因组Ｓｏｕｔｈｅｒｎ杂交分析,表明白色念珠菌中多     

Diversity of Actinobacterial community in saline sediments from Yunnan and Xinjiang, China

The diversity and community structures of actinobacteria in saline sediments collected from Yunnan and Xinjiang Provinces, China, were investigated with cultivation and 16S rRNA gene analysis. A total of 163 actinobacterial isolates were obtained, and they were affiliated with the order Actinomycetales (distributed into five suborders: Streptosporangineae, Micrococcineae, Streptomycineae, Pseudonocardineae, and Glycomycineae). A total of 748 actinobacterial 16S rRNA gene clones were examined, and they could be classified into Actinomycetales, Acidimicrobiales, and unclassified actinobacteria. The Actinomycetales sequences were distributed into nine suborders: Streptosporangineae, Glycomycineae, Micromonosporineae, Pseudonocardineae, Corynebacterineae, Frankineae, Propionibacterineae, Streptomycineae, and Micrococcineae. The unclassified actinobacteria contained three new clusters at the level of subclass or order. Our 16S rRNA gene phylogenetic data indicated that actinobacterial communities were very diverse in the investigated saline sediments (salinity 0.4–11.6%) and some actinobacterial members may be halotolerant or halophilic. The actinobacterial community structures in the saline sediments were different from those in marine and freshwater environments. Our data have implications for a better understanding of the distribution of Actinobacteria in saline environments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identity of an ABA-activated 46 kDa mitogen-activated protein kinase from <Emphasis Type="Italic">Zea mays</Emphasis> leaves: partial purification,identification and characterization

Mitogen-activated protein kinase (MAPK) cascades have been shown to be important components in abscisic acid (ABA) signal transduction pathway. In this study, a 46 kDa MAPK (p46MAPK) induced by ABA was partially purified from maize (Zea mays) by Q-Sepharose FF, Phenyl-Sepharose FF, Resource Q, Mono QTM 5/50 GL, poly-l-lysine-agarose, and Superdex 75 prep-grade columns, and was identified as ZmMAPK5 (gi|4239889) by the matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. Furthermore, the kinase showed optimal activity at pH 8.0, 30°C, and 10 mM MgCl₂; the K _m for myelin basic protein (MBP) substrate and ATP were 0.13 μg μl⁻¹ and 62 μM, respectively. MBP was the preferred substrate, of which the threonine residue was phosphorylated. Finally, the kinase was found to respond to diverse extracelluar stimuli. These results enable us to further reveal the function of the ZmMAPK5 in ABA signaling. The authors Haidong Ding and Aying Zhang contributed equally to the work.     

The complete genome sequence of Bacillus anthracis Ames "Ancestor"

The pathogenic bacterium Bacillus anthracis has become the subject of intense study as a result of its use in a bioterrorism attack in the United States in September and October 2001. Previous studies suggested that B. anthracis Ames Ancestor, the original Ames fully virulent plasmid-containing isolate, was the ideal reference. This study describes the complete genome sequence of that original isolate, derived from a sample kept in cold storage since 1981.     

Improving acidogenic performance in anaerobic degradation of solid organic waste using a rotational drum fermentation system

The effects of leachate from a methanogenic process on acidogenic performance of a solid recycle (SR) process by a rotational drum fermentation (RDF) system were evaluated under mesophilic condition and a hydraulic retention time (HRT) of 20 days. Two SR process configurations, SR1 and SR2, were evaluated, using fresh soybean meal or Okara as substrates. An apparent first-order hydrolysis rate constant of 5.0 x 10(-3)/d for SR1 at pH values of 4.4 and 14.4 x 10(-3)/d for SR2 at pH of 5.0, were obtained. The apparent volatile solids (VS) degradation ratio ranged from 9.6% to 19.4% and total volatile acid (as acetic acid) from 10.8 to 14.9 g/L. Occupying ratios for ionized volatile acid (VA) increased from 30.6% to 63.4% after recycling the leachate to process. However, occupying ratios of acetic acid decreased from 93.3% to 42.0% whereas propionic acid and butyric acid ratios increased in SR2. Integrating the VA production with the hydrolysis rate constants, it is clear that the recirculation of leachate considerably enhanced acidogenic performance of solid recycle process.     

Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from human (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor-binding domain of the S protein being potentially important for SARS progression and tropism. Using the single-round infection assay, we found that a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells. Although single substitution of these two residues had no effects on the infectivity of huSARS-CoV, these recombinant S proteins bound to human ACE2 with different levels of reduced affinity, and the two-amino acid-substituted S protein showed extremely low affinity. On the contrary, substitution of these two amino acid residues of pcSARS-CoV for those of huSRAS-CoV made pcSARS-CoV capable of infecting human ACE2-expressing cells. These results suggest that amino acid residues at position 479 and 487 of the S protein are important determinants for SARS-CoV tropism and animal-to-human transmission.     

Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety

Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.     

Transplantation of somatic nuclei into oocyte cytoplasm reveals that the chromosome properties determine the chromosome separation fate in rabbit

G2/M somatic nuclei were introduced into enucleated meiotically competent oocytes and subsequently cultured in TCM199 plus 10% fetal calf serum (FCS). Pseudo-first polar bodies could be extruded, but the chromosomes failed to arrange normally. Kinetochores were traced with immunofluorescent microscopy using autoimmune sera from patients with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia) scleroderma. In vitro matured oocytes arrested at second meiotic metaphase and kinetochores were detectable as paired structures aligned at the spindle equator. At meiotic anaphase, present or past the kinetochores separated and remained aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis was lost. Kinetochores failed to arrange normally after transferring somatic nuclei into oocytes. Our results suggest that somatic cell nuclei are unable to proceed normally through meiosis when introduced into oocyte meiotic cytoplasm.