Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.     

Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries

We present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Our primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, we adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called "contigs." These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Acidification of small mountain lakes in the High Tatra Mountains,Poland

A hydrochemical investigation was carried out on eight small High Tatra mountain lakes (Poland). When comparing recent data with those from the period 1935–1965, a constant process of acidification of the lakes is found. The average pH of precipitation is 4.8 in the study area, but the lakes are in two stages of acidification: weak (pH 6.0–6.5) and intermediate (pH 5.5–5.8). The differences are due to differences in water sources.     

The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).     

β-1, 3-Glucanase from trichoderma viride. Purification and characterization

The extracellular complex of β-glucanases produced by the mould Trichoderma viride hydrolyzes β-1,3-; β-1,4- and β-1, 6-bonds of β-glucans, as well as mixed β-1,3- and β-1,4-glycosidic linkages. This complex contains also xylanase. The enzymes were isolated from liquid culture medium by centrifuge techniques, concentration and precipitation with acetone. Isolation and purification of β-1,3-glucanase was carried out according to a procedure involving filtration on Bio-Gel P-100, DEAE-Sephadex A 50 and CM-cellulose C-11 chromatography, ultrafiltration and selective adsorption on xylan. The homogeneity of the enzyme was determined by polyacrylamidegel electrophoresis. The purified homogeneous preparation of the isolated β-1,3-glucanase from Tr. riride was subjected to detailed characterization. Amino acid composition, molecular weight and optimum conditions for the enzymatic activity of the protein were determined. The isolated enzyme was shown to be highly specific to substrates with β-1,3-glycosidic linkages; the rate of degradation was found to be proportional to the degree of polymerization of the substrate.     

The production of cellulolytic enzymes by fungal cultures

Summary Twelve fungal cultures belonging to the genera ofAspergillus, Tricboderma, Chaetomium, Stachybotrys, andHypocrea were screened for the production of cellulolytic activity. All twelve were found to degrade xylan, avicel, and carboxymethylcellulose, More cellulolytic activity was obtained with shaken cultures than with still cultures and the addition of citrate-phosphate buffer to the media greatly depressed the levels of cellulolytic activity. Varying the composition of the mineral salts in the medium had no effect on the cellulolytic activity.The growth ofAspergillus wentii under controlled conditions in a bioreactor showed that the cellulolytic activity was not affected by the aeration rate or the type of stirrer. The rate of stirring, however, did effect the cellulolytic activity, as at lower stirring speeds considerable wall growth occurred which resulted in low levels of cellulolytic activity.Culture supernatant fromAspergillus wentii was found to hydrolyze from 30–32% of Solka-Floc and from 2–10% of corn cobs, wheat straw, and newsprint. The extensive hydrolysis of the Solka-Floc indicates that with suitably treated cellulosic wastes and appropriate enzymes, appreciable amounts of sugars could be obtained.     

On the symmetries of multi-palindromic DNA sequences

There are several instances of multiple, overlapping palindromes in DNA sequences which have recently been reported. To ascertain the likelihood of specific biological function for these interdigitating symmetries it is necessary to calculate their probabilities of occurrence. While the probability of occurrence of a single palindrome in a random sequence is calculated in a straightforward fashion, the occurrence of overlapping palindromes is not so simply analyzed. In this paper a general method for handling multiple symmetries is presented. Several theorems concerning the constraints which overlapping symmetries place on each other are presented. A general result is that the probability of occurrence of a symmetry can be significantly enhanced by already existing symmetries. As examples of the theorems and methods developed, the lac operator, the lac CAP-binding site and a region of the λ left operator are examined. The occurrence of several overlapping symmetries appears to be fairly common in such sequences.