Methods for discovering novel motifs in nucleic acid sequences

We describe a computer tool to aid the discovery of new motifsin nucleic acid sequences. A typical use would be to analysea set of upstream regions from a family of related genes inorder to find possible control sequences. The heart of the methodis the creation of dictionaries of related subsequences. Thesedictionaries can then be analysed to look for the commonestor best-defined subsequences, those that occur in the highestnumber of different sequences, or for those in equivalent positionswithin the family. We show the application of the method toa set of E. coli promoter sequences. Received on May 9, 1989; accepted on July 27, 1989     

Metabolic cost of generating muscular force in human walking: insights from load-carrying and speed experiments.

We sought to understand how leg muscle function determines the metabolic cost of walking. We first indirectly assessed the metabolic cost of swinging the legs and then examined the cost of generating muscular force during the stance phase. Four men and four women walked at 0.5, 1.0, 1.5, and 2.0 m/s carrying loads equal to 0, 10, 20, and 30% body mass positioned symmetrically about the waist. The net metabolic rate increased in nearly direct proportion to the external mechanical power during moderate-speed (0.5-1.5 m/s) load carrying, suggesting that the cost of swinging the legs is relatively small. The active muscle volume required to generate force on the ground and the rate of generating this force accounted for >85% of the increase in net metabolic rate across moderate speeds and most loading conditions. Although these factors explained less of the increase in metabolic rate between 1.5 and 2.0 m/s ( approximately 50%), the cost of generating force per unit volume of active muscle [i.e., the cost coefficient (k)] was similar across all conditions [k = 0.11 +/- 0.03 (SD) J/cm3]. These data indicate that, regardless of the work muscles do, the metabolic cost of walking can be largely explained by the cost of generating muscular force during the stance phase.     

A molecular dynamics simulation of a polyamine-induced conformational change of DNA. A possible mechanism for the B to Z transition.

A 75ps molecular dynamics simulation has been performed on a fully solvated complex of spermine with the B DNA decamer (dGdC)5.(dGdC)5. The simulation indicates a possible mechanism by which polyamines might induce the formation of a left-handed helix, the B to Z transition. Spermine was initially located in the major groove, hydrogen bonded to the helix. During the simulation the ligand migrates deeper into the DNA, maintaining strong hydrogen bonding to the central guanine bases and destroying the Watson-Crick base pairing with their respective cytosines. Significant rotation of these and other cytosine bases was observed, in part due to interactions of the helix with the aminopropyl chains of spermine. An intermediate BII conformation might be of importance in this process.     

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.     

The dependence of airway smooth muscle on extracellular Ca2+ for contraction is influenced by the presence of cartilage

Isolated guinea-pig and rabbit airway smooth muscle preparations lacking cartilage are less able to contract, in response to methacholine, histamine and K+, in the absence of extracellular Ca2+ than cartilage-containing preparations removed from the same animal. Cartilage apparently provides utilizable Ca2+ for contraction of airway smooth muscle. The presence of cartilage, therefore, affects the apparent dependence of the isolated smooth muscle on extracellular Ca2+ for contraction.     

Genomic hypomethylation and far-5'' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA