Two types of linkage between codon usage and gene-expression levels

The relation between codon usage and gene-expression levels is an intensively investigated and discussed topic in the field of molecular evolution. We statistically analyzed 25 Escherichia coli gene sequences by a new classification of synonymous codons and found that (i) there are two distinct types of linkage between codon usage and gene-expression levels in E. coli, and (ii) one of the two kinds of codon preferences (the codon preference concerned with interaction of GC/AT choice at three codon positions) is observed significantly in weakly expressed genes.     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.     

Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp. strain NS671.

DNA fragments containing the genes involved in the conversion of 5-substituted hydantoins to their corresponding L-amino acids have been cloned from the 172-kb native plasmid (pHN671) of Pseudomonas sp. strain NS671. The largest recombinant plasmid, designated pHPB14, encoded the ability to convert D-5-substituted hydantoins to the corresponding L-amino acids, whereas the smallest one, designated pHPB12, encoded the ability to convert them to their corresponding N-carbamyl-D-amino acids. Restriction analysis suggested that the inserts of both recombinant plasmids are derived from the identical portion in pHN671 and that the insert of pHPB14, compared with that of pHPB12, has an extra 5.3 kb in length. DNA sequencing revealed that pHPB14 contains two additional complete open reading frames, designated ORF5 and hyuE. Analysis of deletion derivatives of pHPB14 indicated that hyuE is required for the ability to produce L-amino acids from the corresponding D-5-substituted hydantoins, but ORF5 is not. Cells of Escherichia coli transformed with a plasmid containing hyuE were capable of racemizing different 5-substituted hydantoins, indicating that hyuE is a gene encoding a hydantoin racemase.     

Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.     

Tissue distribution of hepatocyte growth factor receptor and its exclusive down-regulation in a regenerating organ after injury.

Using 125I-labeled hepatocyte growth factor (HGF) as a ligand, we examined the tissue distribution of the HGF receptor in adult rats. Specific binding of 125I-HGF was detected in the plasma membranes of liver, spleen, kidney, lung, adrenal gland, pituitary, and thyroid. Scatchard analysis of HGF binding in liver, spleen, kidney, lung, and adrenal gland revealed the presence of a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 400-3,000 sites per ng of plasma membrane protein, the highest number being in the liver. Such a wide distribution of a high affinity HGF receptor indicates that HGF may be a multifunctional growth factor, targeting to a variety of organs, and not restricted to liver. After 70% partial hepatectomy, specific binding of 125I-HGF to membranes of the residual liver rapidly decreased, but there was no change in the kidney, lung, and spleen. On the other hand, after unilateral nephrectomy rapid down-regulation of the HGF receptor was clearly evident in the remaining kidney, but not in other organs including the liver. These findings suggest the presence of control mechanisms governing HGF receptor function only in a regenerating organ after injury.     

Proportional hazards model with covariates subject to measurement error.

When covariates of a proportional hazards model are subject to measurement error, the maximum likelihood estimates of regression coefficients based on the partial likelihood are asymptotically biased. Prentice (1982, Biometrika 69, 331-342) presents an example of such bias and suggests a modified partial likelihood. This paper applies the corrected score function method (Nakamura, 1990, Biometrika 77, 127-137) to the proportional hazards model when measurement errors are additive and normally distributed. The result allows a simple correction to the ordinary partial likelihood that yields asymptotically unbiased estimates; the validity of the correction is confirmed via a limited simulation study.     

Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system.

Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.     

Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.     

R1-20, a novel monoclonal antibody reacting with a molecule distinct from integrin family, induces homotypic cell aggregation.

R1-20, a novel mAb reacting with a cell surface Ag on normal human lymphocytes and leukemic cell lines, was shown to induce homotypic cell aggregation in leukemic cell lines. This phenomenon was specific to mAb R1-20 because antibodies recognizing CD2, CD7, CD28, and HLA-ABC failed to exhibit homotypic cell aggregation. Induction of aggregation by mAb R1-20 occurred at 37 degrees C, but not at 4 degrees C and required cytoskeletal integrity. Sodium azide, a metabolic inhibitor, had no effect on the aggregation. Distinct from lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction in which divalent cations are essential elements, R1-20-mediated aggregation was not abolished with EDTA treatment. The R1-20 Ag was determined as a molecule of M(r) 100 to 110 kDa in immunoprecipitation and immunoblotting methods, under both reducing and nonreducing conditions. The molecular composition is quite different from that of any known integrin molecule. The R1-20 Ag was expressed on resting and activated T Lymphocytes as well as on normal B lymphocytes. Monocytes and granulocytes had no detectable R1-20 Ag. Among the leukemia-derived cell lines we used, mAb R1-20 reacted with 18 of 32 T cell lines, 2 of 20 B cell lines, 2 of 3 non-T-non-B cell lines, 2 of 7 myelomonocytic cell lines, and 2 of 3 nonlymphoid-nonmyeloid cell lines. All EBV-transformed B cell lines examined (10 cell lines) were R1-20+. The spectrum of reactivity among the cell lines tested was different from that of known antiadhesion antibodies tested. All these findings indicate that the Ag recognized by mAb R1-20 may represent a new type of cell adhesion molecule.