Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum

The complete nucleotide sequence of the celH gene of Clostridium thermocellum was determined. The open reading frame extended over 2.7-kb DNA fragment and encoded a 900-amino acid (aa) protein (Mr 102,301) which hydrolyzes carboxymethylcellulose, p-nitrophenyl-beta-D-cellobioside, methylumbelliferyl- beta-D-cellobioside, barley beta-glucan, and larchwood xylan. The N terminus showed a typical signal peptide, and a cleavage site after Ser44 was predicted. Two Pro-Thr-Ser-rich regions divided the protein into three approximately equal domains. The central 328-aa region was similar to the N-terminal part, carrying the active site, of C. thermocellum endoglucanase E (EGE; 30.2%). The C-terminal region ended with two conserved 24-aa stretches showing close similarity with those previously described in EGA, EGB, EGD, EGE, EGX, and xylanase from C. thermocellum. Deletions of celH removing up to 327 codons from the 5' end and up to 245 codons from the 3' end of the coding sequence did not affect enzyme activity, confirming that the central domain was indeed responsible for catalytic activity. Production of truncated EGH in Escherichia coli was increased up to 120-fold by fusing fragments containing the 3' portion of the gene with the start of lacZ' present in pTZ19R.     

A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli

Abstract Two independent collections of clones containing Clostridium thermocellum genes involved in cellulose have been previously obtained at IAPGR, Cambridge, and at the Pasteur Institute, Paris. The two collections were compared for cross-hybridization, restriction maps and enzyme phenotypes. Truly distinct genes were one β-glucosidase gene, two xylanase genes, and fifteen endogluconase genes. Two of the cloned fragments contained extraneous DNA which was absent from their respective counterparts isolated in the other collection. The dicrepancies resulted from in vivo rearrangements which had occurred in either of the C. thermocellum NCIB 10682 stocks used to generate the two gene banks.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum

Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.