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Mutational Analysis of the Myxococcus xanthus  4499
Promoter Region Reveals Shared and Unique Properties in Comparison with Other
C-Signal-Dependent Promoters.Deborah R. Yoder, 2004.The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior . C-signal affects gene expression late in development, including that of 4499,
an operon identified by insertion of Tn5 lac into the M .
xanthus chromosome . The
4499
promoter region has several sequences in common with those found
previously to be important for expression of other C-signal-dependent
promoters . To determine if these sequences are important for
4499
promoter activity, the effects of mutations on expression of a
downstream reporter gene were tested in M . xanthus . Although
the promoter resembles those recognized by Escherichia coli
 54,
mutational analysis implied that a
70-type
factor likely recognizes the promoter . A 7-bp sequence known as a C
box and a 5-bp element located 6 bp upstream of the C box have been
shown to be important for expression of other C-signal-dependent
promoters . The
4499
promoter region has C boxes centered at –33 and –55 bp, with 5-bp
elements located 7 and 8 bp upstream, respectively . A
multiple-base-pair mutation in any of these sequences reduced
4499
promoter activity more than twofold . Single base-pair mutations in
the C box centered at –33 bp yielded a different pattern of effects
on expression than similar mutations in other C boxes, indicating
that each functions somewhat differently . An element from about –81
to –77 bp exerted a twofold positive effect on expression but
did not appear to be responsible for the C-signal dependence of the
4499
promoter . Mutations in sigD and sigE, which are genes
that encode
factors, reduced expression from the
4499
promoter . The results provide further insight into the regulation
of C-signal-dependent genes, demonstrating both shared and unique
properties among the promoter regions so far examined .
Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336. L. Dudley Eirich, 2004.Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of  -hydroxy fatty acids to
-aldehydes
by a fatty alcohol oxidase (FAO) . Three FAO genes (one gene
designated FAO1 and two putative allelic genes designated
FAO2a and FAO2b), have been cloned and sequenced from
this strain . A comparison of the DNA sequence homology and derived
amino acid sequence homology between these three genes and previously
published Candida FAO genes indicates that
FAO1 and FAO2 are distinct genes . Both genes were
individually cloned and expressed in Escherichia coli . The
substrate specificity and Km values for
the recombinant FAO1 and FAO2 were significantly different.
Particularly striking is the fact that FAO1 oxidizes
-hydroxy
fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but
not
-hydroxy fatty acids . Analysis of extracts of strain H5343
during growth on fatty acids indicated that only FAO1 was highly
induced under these conditions . FAO2 contains one CTG codon,
which codes for serine (amino acid 177) in C . tropicalis but
codes for leucine in E . coli . An FAO2a construct,
with a TCG codon (codes for serine in E . coli) substituted for
the CTG codon, was prepared and expressed in E . coli . Neither
the substrate specificity nor the Km
values for the FAO2a variant with a serine at position 177 were
radically different from those of the variant with a leucine at that
position .
Trehalose-Mediated Inhibition of the Plasma Membrane H+-ATPase from Kluyveromyces lactis: Dependence on Viscosity and Temperature. José G. Sampedro, 2002.The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures . At 20°C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased Vmax and increased S0.5 (substrate concentration when initial velocity equals 0.5 Vmax), mainly at high trehalose concentrations (0.6 to 0.8 M) . The quotient Vmax/S0.5 decreased from 5.76 µmol of ATP mg of protein-1 min-1 mM-1 in the absence of trehalose to 1.63 µmol of ATP mg of protein-1 min-1 mM-1 in the presence of 0.8 M trehalose . The decrease in Vmax was linearly dependent on solution viscosity (  ), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution . In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted . In the absence of trehalose, increasing the temperature up to 40°C resulted in an exponential increase in Vmax and a decrease in enzyme cooperativity (n), while S0.5 was not modified . As temperature increased, the effect of trehalose on Vmax decreased to become negligible at 40°C, in good correlation with the temperature-mediated decrease in viscosity . The trehalose-mediated increase in S0.5 was similar at all temperatures tested, and thus, trehalose effects on Vmax/S0.5 were always observed . Trehalose increased the activation energy for ATP hydrolysis . Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock .
Rates and Consequences of Recombination between rRNA Operons. Joel G. Hashimoto, 2003.A mutant strain of Escherichia coli was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (sacB-neo) into the small-subunit rRNA-encoding gene rrs in the rrnB operon . During growth in a complex medium, the cassette was lost from the population, and a complete rrs gene was restored at a rate of 5 x 10-9 per cell division . Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons . The relative fitness of strains with restored rrnB operons was 1 to 2% higher than that of the mutant strain . The rrnB operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in rrnC, -E, and -G . In 2 of the 14 strains in which rrnB was restored, the spacer region had the same length as the spacer region in rrnA, -D, and -H . The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of rrnB suggest that the restoration was accomplished via gene conversion . The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in E . coli . These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes .
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