The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Antisuppression by mutations in elongation factor Tu

Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild-type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.     

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles

Summary We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage receptor protein in its outer membrane. Phage adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using vehicles carrying transposons. This system can also be used for cosmid cloning.     

Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli.

Escherichia coli K-12 minicells were employed to investigate the biosynthesis of plasmid-encoded, heat-labile enterotoxin of E. coli. Two polypeptide species related to the B subunit of the toxin were expressed in the minicells. One of these polypeptides (molecular weight, 11,500) was immunoprecipitated by antiserum to cholera toxin. Because the B subunits of heat-labile enterotoxin and cholera toxin have common antigenic sites, we concluded that this species was the mature B subunit. The larger polypeptide (molecular weight, 13,000) is likely to be a precursor of the B subunit because it could be chased into the mature form. This conversion was inhibited by compounds which dissipate proton motive force, suggesting that processing requires energy.     

Bacteriological Quality of Raw Materials Used in Finnish Mink Feed

Mink feed raw materials were analyzed for total bacterial count, the number of faecal streptococci, the coliform count, the number of haemolytic bacteria and the number of sulphite reducing bacteria. The investigation comprised samples from the following raw materials: four slaughter-house offal products, preserved and unpreserved slaughter blood, Baltic herring, cod filletting offal, fish silage, blood meal, fish meal, meat-bone meal, protein concentrate, brewer’s yeast and cereal feed. The slaughter-house offals and unpreserved slaughter blood had the poorest quality, in terms of all the bacterial types for which the samples were analyzed. There were statistically significant differences in bacterial contents between slaughter-house offals from different sources. The preserved slaughter blood had significantly lower bacterial contents as compared to the unpreserved slaughter blood. Single samples of the cod filletting offal, Baltic herring and the blood meal had relatively high total bacterial counts, but the specified mean bacterial counts were relatively low. The bacterial counts for the rest of the investigated raw materials were relatively low.     

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose.

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

Arrangement of protein I in Escherichia coli outer membrane: cross-linking study.

The arrangement of protein I in the outer membrane of Escherichia coli was investigated by cross-linking whole cells, isolated cell wall, protein-peptidoglycan complexes, and protein I released from peptidoglycan with NaCl. Both cleavable azide cross-linkers and imidoester reagents were used. The data presented suggest that protein I exists in the outer membrane as a trimer.     

Leukemia Prediction Using Sparse Logistic Regression

We describe a supervised prediction method for diagnosis of acute myeloid leukemia (AML) from patient samples based on flow cytometry measurements. We use a data driven approach with machine learning methods to train a computational model that takes in flow cytometry measurements from a single patient and gives a confidence score of the patient being AML-positive. Our solution is based on an regularized logistic regression model that aggregates AML test statistics calculated from individual test tubes with different cell populations and fluorescent markers. The model construction is entirely data driven and no prior biological knowledge is used. The described solution scored a 100% classification accuracy in the DREAM6/FlowCAP2 Molecular Classification of Acute Myeloid Leukaemia Challenge against a golden standard consisting of 20 AML-positive and 160 healthy patients. Here we perform a more extensive validation of the prediction model performance and further improve and simplify our original method showing that statistically equal results can be obtained by using simple average marker intensities as features in the logistic regression model. In addition to the logistic regression based model, we also present other classification models and compare their performance quantitatively. The key benefit in our prediction method compared to other solutions with similar performance is that our model only uses a small fraction of the flow cytometry measurements making our solution highly economical.