Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

Measurement of double-strand breaks in Chinese hamster cell DNA by neutral filter elution: calibration by 125I decay

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.     

Filter elution assays for DNA damage: practical and mechanistic significance of the DNA in the filter support wash.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.     

Assessment of the biological stability of human thyroid stimulating hormone prepared by immunoaffinity chromatography

This paper reports a comprehensive study of the biological stability of an immunoaffinity purified preparation of human thyroid stimulating hormone (TSH) and provides a reference against which future natural or synthetic preparations may be compared. The stability of the hormone preparation was investigated using the accelerated degradation method. The bioassay of the TSH was carried out in mice using a modified McKenzie method. Analysis of the results showed that the preparation was as stable as other TSH preparations purified by conventional methods.     

Selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity

The role of adhesive selectin molecules in the process of atherogenesis is an open question. These molecules are known as markers of atherosclerosis activity, however, only some biological mechanisms are known up to now. In this study we examined the levels of soluble forms of E-, P-selectin and monocyte chemotactic protein (MCP-1) in the process of extracorporeal cholesterol elimination by LDL-apheresis. We measured the levels of sE-, sP-selectin and MCP-1 in the plasma before and after LDL-apheresis and in the washout solution from immunoabsorption columns Lipopak. Eighty measurements were performed repeatedly in 6 patients with severe familial hypercholesterolemia (FH) on long-term LDL-apheresis treatment. Before the procedure P-selectin levels were 204+/-179 ng/ml, E-selectin 32.1+/-33.7 ng/ml, MCP-1 323.8+/-121 pg/l, whereas after the procedure we found P-selectin levels 131.6+/-34 ng/ml, E-selectin 33.1+/-51 ng/ml, and MCP-1 200.4+/-15 pg/l. Levels of P-selectin were increased in the blood of patients with FH in spite of long-term intensive extracorporeal LDL-elimination, documenting thus the activity of atherosclerosis. The levels of P-selectin and MCP-1 decreased significantly after the hypolidemic procedure and could be used as another marker showing the effectivity of the extracorporeal LDL-cholesterol elimination (immediately after the procedure), and, after further verification, may serve as a marker for controlling the therapy efficacy.     

Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.     

New insights on the mechanism of acid degradation of pea starch

The degradation of pea starch granules by acid hydrolysis has been investigated using a range of chemical and structural methods, namely through measuring changes in amylose content by both the iodine binding and concanavalin A precipitation methods, along with small angle X-ray scattering (SAXS), wide angle X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). The relative crystallinity, intensity of the lamellar peak and the low-q scattering increased during the initial stages of acid hydrolysis, indicating early degradation of the amorphous regions (growth rings and lamellae). In the first 2 days of hydrolysis, there was a rapid decline in amylose content, a concomitant loss of precipitability of amylopectin by concanavalin A, and damage to the surface and internal granular structures was evident. These observations are consistent with both amylose and amylopectin being located on the surface of the granules and attacked simultaneously in the early stages of acid hydrolysis. The results are also consistent with amylose being more concentrated at the core of the granules. More extensive hydrolysis resulted in the simultaneous disruption of amorphous and crystalline regions, which was indicated by a decrease in lamellar peak intensity, decrease in interhelix peak intensity and no further increase in crystallinity. These results provide new insights into the organization of starch granules.