Verner L. Seligy

The Glu residue in the conserved ASN-Glu-Pro sequence of two highly divergent endo-β-1,4-glucanases is essential for enzymatic activity

We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.

Complete amino acid sequence of goose erythrocyte H5 histone and the homology between H1 and H5 histones

Summary Goose H5 histone has 193 amino acid residues and a molecular weight of 20,903. Residues 1–28 and 101–193 are probably extended while residues 29–100 are in a globular conformation. An alignment of availalbe sequences from other lysine-rich histones indicates H5 species variability is limited mostly to the N-terminal domain whereas variability between H1 and H5 proteins is within both N and C terminal domains. The constancy in size of the central domain may be important for the function of all lysine-rich histones.

Binding of ethidium bromide to avian erythrocyte chromatin

Summary Ethidium bromide intercalation studies indicate that mature avian erythrocyte chromatin has 5.6 times fewer primary binding sites than deproteinized DNA. The number of both primary and secondary binding sites was increased upon removal of chromosomal proteins; among those, removal of avian erythrocyte specific histone V shows the highest increment in dye binding.

The large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase is encoded in the inverted repeat sequence of the Chlamydomonas eugametos chloroplast genome

SummaryThe physical mapping of Aval, BstEII and EcoR1 restriction sites on the chloroplast genome of the green alga Chlamydomonas eugametos is presented. The circular map, with a size of 243 kilobase pairs, is the largest yet reported for a chloroplast genome. It features a large inverted repeat sequence, part of which encodes the 16S and 23S ribosomal RNAs (rRNAs), the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and the “32-kdodalton” thylakoid membrane protein (psbA). Such an rRNA-encoding inverted repeat sequence is also found in the chloroplast genomes of Chlamydomonas reinhardtii and most land plants. These genomes, however, differ from that of C. eugametos by the absence of the rbcL gene from the inverted repeat sequence of C. reinhardtii and by the absence of both the rbcL and psbA genes from the inverted repeat sequence of land plants. Possible evolutionary implications of these differences are discussed.

Amino acid sequence homologies between H1 and H5 histones.

Abstract The amino acid sequence of the first 112 residues of goose H5 and the first 38 residues of pigeon H5 has been determined. When a gap of 12 residues is introduced in the N-terminal regions of avian H5, almost half of the residues become identical to those of H1 from various species. The region of this gap corresponds to a homologous repeating sequence in H1. This suggests a deleation or duplication in this region and that the genes for H1 and H5 histones have evolved from a common ancestral gene.

Species variability of N-terminal sequence of avian erythrocyte-specific histone H5

Abstract A comparison of the N-terminal amino acid sequence of H5 isolated from chicken, quail, duck, goose and pigeon shows considerable sequence variation. With the possible exception of the very lysine-rich histone H1, H5 variation is much more extensive than that reported previously for other histones. The observed sequence diversity is in agreement with the known taxonomic grouping of these birds.

Glucanase gene diversity in prokaryotic and eukaryotic organisms.

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.

Multiplicity and distribution of rDNA cistrons among chromosome I and VII aneuploids of Saccharomyces cerevisiae

Abstract The strains characterized in this study were simple (trisomy or tetrasomy for chromosome I or VII, i.e. 2n + 1I or 2n + 2I or 2n + 1VII or 2n + 2VII) and double (2n + 1I + 1VII, 2n + 2I + 1VII and 2n + 2I + 2VII) aneuploids. All aneuploids were essentially homozygous and identical in their genetic background of the 15 chromosomes other than I or VII. In addition to observed differences in colonial morphologies (ref. [2] and this study table 1) the simple aneuploids were found to exhibit depressed growth rates and possibly diminished amounts of rRNA (26s + 17s + 5s) relative to either the normal diploid or the double aneuploids. The rRNA (26s + 17s) was purified electrophoretically and used with the technique of RNA-DNA hybridization to determine the total content of rRNA genes and the relative distribution of these genes in the aneuploids. The annealing studies confirmed previous reports using monosomic strains of S. cerevisiae [4–6] that about two-thirds or more of the rRNA genes are located on chromosome I. The results further show that no more than 10–15% of the remaining rRNA genes could be assigned to chromosome VII. These results suggest that the phenotypic changes associated with the inclusion of extra copies of chromosome VII are due to factors other than a simple increase in rRNA genes.

Distribution of intercalative dye binding sites in chromatin

Actinomycin D (AMD) and ethidium bromide (EB) were found to bind to chromatin isolated from a variety of gander tissues according to a strong and weak process analogous to that found for deproteinized DNA. Distribution of the dye intercalation sites in chromatin and DNA were evaluated at low r-values (dye bound per nucleotide) by following the appearance of free dye released from chromatin and DNA during thermal denaturation. The AMD dissociation profiles closely resembled the DNA or chromatin-DNA denaturation profiles; whereas the EB derivative dissociation profiles, indicated 3 major transitions for transcriptionally active chromatin with the main component corresponding to the single component which characterizes DNA. The DNA-like component was greatly reduced for mature erythrocyte chromatin but could be generated by removal of histone I and V. Removal of residual non acid-soluble proteins from dehistonized chromatin, urea treatment or dissociation and reconstitution of chromatin favoured conversion to the DNA-like component with loss of the other two. This study indicates that more than one type of binding exists generally in chromatin.