基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.     

Leukocyte deformability: finite element modeling of large viscoelastic deformation.

An axisymmetric deformation of a viscoelastic sphere bounded by a prestressed elastic thin shell in response to external pressure is studied by a finite element method. The research is motivated by the need for understanding the passive behavior of human leukocytes (white blood cells) and interpreting extensive experimental data in terms of the mechanical properties. The cell at rest is modeled as a sphere consisting of a cortical prestressed shell with incompressible Maxwell fluid interior. A large-strain deformation theory is developed based on the proposed model. General non-linear, large strain constitutive relations for the cortical shell are derived by neglecting the bending stiffness. A representation of the constitutive equations in the form of an integral of strain history for the incompressible Maxwell interior is used in the formulation of numerical scheme. A finite element program is developed, in which a sliding boundary condition is imposed on all contact surfaces. The mathematical model developed is applied to evaluate experimental data of pipette tests and observations of blood flow.     

Baculovirus phosphoprotein pp31 is associated with virogenic stroma.

The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.     

Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli.

Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.     

Induction of F9 cell differentiation by transient exposure to retinoic acid.

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.     

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15

The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.