Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.     

Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi

In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi     

利用带有原核增强子样序列的新型载体在大肠杆菌中高效表达人α肿瘤坏死因子

将去除信号肽的人肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)cDNA插入到带有原核增强子样序列Px的新型表达载体pBV320中,使TNF cDNA 5′端直接置于大肠杆菌trp启动子下游,采用37℃恒温培养,使TNF在大肠杆菌中获得了高效表达,表达活性达1.35(±0.17)×10~6U/L菌液。表达的TNF-α对L929细胞的毒性作用可被抗人肿瘤坏死因子-α的单克隆抗体所中和。表达菌裂解液作SDS-聚丙烯酰胺凝胶电泳,显示有一条分子量与TNF分子量吻合、约为17000道尔顿的蛋白带。利用DEAE-Sepharose阴离子交换层析及Sephacryl S-200凝胶过滤对上述重组人TNF-α进行纯化,获得电泳纯产品,比活性为1.48×10~6U/mg。     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (FA) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Fetal and variant alpha-fetoproteins are encoded by mRNAs that differ in sequence at the 5' end

The temperature-sensitive RLA209-15 fetal rat hepatocyte line grown at the nonpermissive temperature (40 degrees C, normal phenotype) produces authentic rat alpha-fetoproteins (AFPs) of 69K and 73K (fetal AFPs) which are encoded by a 2.2-kb mRNA. These cells also produce low levels of a 1.7-kb AFP mRNA and a 65K variant AFP when grown at the permissive temperature (33 degrees C, transformed phenotype). Hybrid-selected translation demonstrates that the 1.7-kb AFP mRNA encodes the 65K variant AFP. Northern blot hybridization and S1 nuclease analyses indicate that the 1.7-kb mRNA lacks sequences present in the first seven 5' exons of the 2.2-kb AFP mRNA. However, the 1.7- and 2.2-kb AFP mRNAs share common sequences extending from the beginning of the eighth exon (corresponding to nucleotide 873 of the fetal AFP mRNA) to the 3' end. Primer extension analysis suggests that the 1.7-kb RNA contains additional sequences 5' to the common regions shared by both AFP mRNAs. We have previously shown that adult rat liver produces a 1.7-kb AFP mRNA; we now report the isolation of a cDNA (ARFP5) encoding this variant AFP mRNA from an adult rat liver cDNA library. Restriction endonuclease mapping and sequence analysis of ARFP5 confirm that the 1.7- and 2.2-kb AFP mRNAs share similar sequences at the 3' region (approximately 1.1 kb). However, ARFP5 contains an additional 90 bp variant AFP mRNA-specific 5' sequence which is located in the seventh intron of the rat AFP gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of DNA sequence on the nature of mispairing during DNA synthesis

A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template. The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates. The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template. Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing. The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template. However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.     

Aggressive chemotherapy in adult primary myelodysplastic syndromes. A report on 29 cases

Twenty-nine adult patients with primary myelodysplastic syndromes (MDS) and an excess of marrow blasts were treated by aggressive chemotherapy while still in MDS phase (20 cases) or after progression to ANLL (9 cases). Median age was 47.5 (range 18-68). Twenty-eight patients received a combination of Rubidazone and Ara C and 1 received High dose Ara C. Fourteen patients (48%) achieved complete remission (CR), 5 (17%) were treatment failures (F) and 10 (35%) died during therapy induced aplasia (DA). Median disease free survival was 8.5 months. Median survival of the whole population was 6 months from the onset of treatment, and 17 months in patients achieving CR. These results were significantly less favorable than those obtained at our institution in de novo ANLL with the same chemotherapy regimens. No statistically significant prognostic factors of treatment outcome emerged but patients with normal cytogenetic findings seemed to have both a higher CR rate and longer remissions than patients with abnormal karyotypes. Patients under 50 did not have higher CR rates than older patients, although they had longer remissions (with 3 out of 6 CRs exceeding 2 years). Finally, treatment outcome and survival were identical in patients treated in the MDS phase and in those treated after progression to ANLL. Combination chemotherapy is a highly toxic approach in MDS and essentially seems to benefit younger patients with a normal karyotype, in whom some long remissions can be obtained.