Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA.

Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter.     

Ratio of the mutation spectrum to the physiological state of the Penicillium chrysogenum cell. Auxotrophic mutations induced by UV rays at different stages of DNA replication

The spectrum of the mutations obtained with exposure of nonactivated spores of Pen. chrysogenum to N-nitrozo-N-methylbiuret (NMB) was studied and it was shown that the mutations associated with blocking of amino acid synthesis predominated. When activated spores of Pen. chrysogenum were treated with the mutagen, correlation was observed between the spectrum of the induced mutations, the physiological state of the cells and the exposure time. It is supposed that the mutagen molecule affects the replicating part of the DNA molecule. Schemes for predominating liberation of the mutations according to the markers in time were developed. This may give an idea of the order of the markers in genome.     

Characterization and suppression of dysfunctional human alpha1-antitrypsin variants

Human alpha1-antitrypsin-deficient variants may aggregate in the liver, with subsequent deficiency in the plasma, which can lead to emphysema. The structural and functional characteristics of 10 dysfunctional alpha1-antitrypsin variants (R39C, S53F, V55P, I92N, G115S, N158K, E264V, A336T, P369S, and P369L) were analyzed in detail. Most of them were unstable, as compared to the wild-type molecule, and many of the variants folded into an intermediate form. When five thermostable mutations (T68A, A70G, M374I, S381A, and K387R) were introduced into dysfunctional alpha1-antitrypsin variants, the stabilities and inhibitory activities of most of the variants were restored to levels comparable to those of the wild-type molecule. However, the extremely unstable S53F variant was not stabilized sufficiently by these mutations so as to exhibit function. N158K variant, which carries a mutation in the region critical for the reactive site loop insertion into beta-sheet A, exhibited a reduced level of inhibitory activity, despite conformational stabilization. These results show that aberrant folding caused by conformational destabilization due to mutations can be compensated for by increasing the overall stability of the alpha1-antitrypsin molecule, with exception of a mutation in the highly localized region critical for functional execution.     

Mutational analysis of bacteriophage lambda lysis gene S

A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.     

Derepression of F factor function in Salmonella typhimurium

In Salmonella typhimurium LT2 the F factor of Escherichia coli K-12 replicates normally but is repressed; Flac+ cells give no visible lysis on solid media with male-specific phages, low frequency transfer of Flac+ (0.001-0.007 per donor cell), few f2 infective centers (0.002-0.006 per cell), and they propagate male-specific phages to low titers. Thus they display a Fin+ (fertility inhibition) phenotype. This repression, owing to pSLT, a 60 Mdal plasmid normally resident in S. typhimurium, was circumvented by the following materials: (i) Flac+ plasmids from E. coli with mutations in finP or traO; (ii) a S. typhimurium line which had been cured of pSLT; (iii) pKZl, a KmR plasmid in the same Inc group as pSLT, which caused expulsion of pSLT and made Fin- lines; (iv) F-Fin- mutants which originated spontaneously and which are present in most Hfr strains of S. typhimurium. Strains which are derepressed for F function by the above methods give visible lysis on solid media with male-specific phages, ca. 1.0 Lac+ recombinants per donor cell in conjugal transfer, ca. 0.82 f2 infective centers per cell, over 80% of cells with visible F pili, and propagation of male-specific phages to high titer. These data confirm earlier observations that pSLT represses F by the FinOP system. In addition, it shows that there is no other mechanism which represses F function in S. typhimurium. If donor function is derepressed by one of the above methods, and if rough recipient strains are used, F-mediated conjugation in S. typhimurium LT2 is as efficient as in E. coli K-12.     

Sequence analyses of presenilin mutations linked to familial Alzheimer’s disease

Familial Alzheimer’s disease (FAD)-linked presenilin (PS) mutations show gain-of-toxic-function characteristics. These FAD PS mutations are scattered throughout the PS molecule, reminiscent of the distribution of cystic fibrosis transmembrane conductance regulator and p53 mutations. Because of the scattered distribution of PS mutations, it is difficult to infer mechanistic insights about how these mutations cause the disease similarly. Recent careful reexamination of γ-secretase activity indicates that some PS mutations decrease the proteolytic activity of γ-secretase, suggesting a loss-of-function nature of PS mutations. To extend this observation to all known PS mutations, a large number of PS mutations were evaluated using bioinformatic tools. The analyses reveal that as many as one third of PS1 residues are highly conserved, that about 75% of FAD mutations are located to the highly conserved residues, and that most PS mutations likely damage the activity of PS. These results are consistent with the idea that the majority of PS mutations lower the activity of PS/γ-secretase.     

A 3D model of human P450c21: study of the putative effects of steroid 21-hydroxylase gene mutations

In order to better understand the disease-causing role of missense mutations found in the CYP21 gene from patients affected with congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we built two three-dimensional (3D) models of human P450c21 using all known 3D structures of P450s. For each residue affected by a missense mutation, its location in the 3D structure and the putative changes in terms of biochemical properties brought about by the mutation were analyzed. Most of the severe alleles were found to affect residues located in functionally important regions of the molecule such as substrate recognition sites (SRS) or the heme region, whereas moderate mutations were mostly found in less crucial regions of the molecule. Thus, there is a good correlation between the 3D structure study and clinical data and mutagenesis experiments previously reported. In one case, however, the observed clinical severity of the mutation (E380D) did not match its expected severity deduced from the model, pointing to a potential functionally important region of the molecule. Our 3D human models provide a basic model for further studies of mutations responsible for 21-hydroxylase, and for identification of important residues involved in the specific activity of the enzyme.     

lambda-Transducing phages carrying transfer genes isolated from an abnormal prophage insertion into the traY gene of F

A set of lambda-transducing phages carrying transfer (tra) genes has been isolated from an abnormal lysogen in which a lambda prophage was inserted into the traY gene of Flac. These have been characterized genetically for complementation of Flac tra and finP point mutants and for the presence of oriT. Studies of tra gene expression during lambda repression showed that tra genes on the transducing phages were expressed from the lambda PL promoter as well as from the transfer promoters when these were present. The molecular weights of the traM (14,000) and traJ (23,500) proteins were measured after infection of ultraviolet-irradiated cells with one of the phages, ED lambda 102, and overproduction of the traJ protein upon induction of an ED lambda 102 lysogen was demonstrated. A proportion of this traJ protein was located in the inner membrane and cytoplasmic fractions of the cell, the majority being in the outer membrane. Physical analysis of the DNA carried by the lambda tra phages by determination of the phage buoyant densities in CsCl, by restriction enzyme digestion and by electron microscope heteroduplex analysis, was used to define the DNA segments encoding the tra functions. Correlation of the physical and genetical data improved the positioning of the tra genes within the transfer region. These results were combined with new restriction enzyme cleavage data to construct an improved map of this region.     

Distribution of the native strain in human alpha 1-antitrypsin and its association with protease inhibitor function

Serine protease inhibitors (serpins) are metastable in their native state. This strain, which is released upon binding to target proteases, is essential for the inhibitory activity of serpins. To understand the structural basis of the native strain, we previously characterized stabilizing mutations of alpha(1)-antitrypsin, a prototypical inhibitory serpin, in regions such as the hydrophobic core. The present study evaluates the effects of single point mutations throughout the molecule on stability and protease inhibitory activity. We identified stabilizing mutations in most secondary structures, suggesting that the native strain is distributed throughout the molecule. Examination of the substitution patterns and the structures of the mutation sites revealed surface hydrophobic pockets as a component of the native strain in alpha(1)-antitrypsin, in addition to the previously identified unusual interactions such as side chain overpacking and cavities. Interestingly, many of the stabilizing substitutions did not affect the inhibitory activity significantly. Those that affected the activity were confined in the regions that are mobilized during the complex formation with a target enzyme. The results of our study should be useful for designing proteins with strain and for regulating the stability and functions of serpins.     

Specific duplications fostered by a DNA structure containing adjacent inverted repeat sequences

This paper describes the nucleotide sequences of three spontaneous mutations in a suppressor gene of phage T4 tRNA(Ser). They are duplications of the anticodon and variable arms of the tRNA(Ser) molecule. One is a 34-nucleotide direct repeat of the wild-type sequence. The remaining two have reciprocal structures, with each containing 35-nucleotide inverted and direct repeats of the wild-type sequence. One of the latter mutations is frequent and was present in multiple isolates. All three duplications are unstable, and several revertants of each were sequenced. Most of the revertants had the wild-type nucleotide sequence; however, one had imprecisely removed the duplicated residues, leaving four new nucleotides compared to the wild-type sequence. These mutations represent significant genetic events with regard to their high rates and their gross structural alterations. As to their origin, the mutations can be described as the end-products of endonuclease cleavage of DNA at regions of potential secondary structure and subsequent DNA synthesis. The secondary structure contains four base-paired stems that emerge from duplex DNA. These stems encode the anticodon and variable arm regions of the tRNA(Ser) molecule. The cleavage sites mimic the known substrate of T4 endonuclease VII, an enzyme previously noted for its ability to resolve Holliday-like DNA intermediates.     

Uncovering the profile of somatic mtDNA mutations in Chinese colorectal cancer patients

In the past decade, a high incidence of somatic mitochondrial DNA (mtDNA) mutations has been observed, mostly based on a fraction of the molecule, in various cancerous tissues; nevertheless, some of them were queried due to problems in data quality. Obviously, without a comprehensive understanding of mtDNA mutational profile in the cancerous tissue of a specific patient, it is unlikely to disclose the genuine relationship between somatic mtDNA mutations and tumorigenesis. To achieve this objective, the most straightforward way is to directly compare the whole mtDNA genome variation among three tissues (namely, cancerous tissue, para-cancerous tissue, and distant normal tissue) from the same patient. Considering the fact that most of the previous studies on the role of mtDNA in colorectal tumor focused merely on the D-loop or partial segment of the molecule, in the current study we have collected three tissues (cancerous, para-cancerous and normal tissues) respectively recruited from 20 patients with colorectal tumor and completely sequenced the mitochondrial genome of each tissue. Our results reveal a relatively lower incidence of somatic mutations in these patients; intriguingly, all somatic mutations are in heteroplasmic status. Surprisingly, the observed somatic mutations are not restricted to cancer tissues, for the para-cancer tissues and distant normal tissues also harbor somatic mtDNA mutations with a lower frequency than cancerous tissues but higher than that observed in the general population. Our results suggest that somatic mtDNA mutations in cancerous tissues could not be simply explained as a consequence of tumorigenesis; meanwhile, the somatic mtDNA mutations in normal tissues might reflect an altered physiological environment in cancer patients.     

Multi-targeting of K-Ras domains and mutations by peptide and small molecule inhibitors

K-Ras activating mutations are significantly associated with tumor progression and aggressive metastatic behavior in various human cancers including pancreatic cancer. So far, despite a large number of concerted efforts, targeting of mutant-type K-Ras has not been successful. In this regard, we aimed to target this oncogene by a combinational approach consisting of small peptide and small molecule inhibitors. Based on a comprehensive analysis of structural and physicochemical properties of predominantly K-Ras mutants, an anti-cancer peptide library and a small molecule library were screened to simultaneously target oncogenic mutations and functional domains of mutant-type K-Ras located in the P-loop, switch I, and switch II regions. The selected peptide and small molecule showed notable binding affinities to their corresponding binding sites, and hindered the growth of tumor cells carrying K-Ras^G12D and K-Ras^G12C mutations. Of note, the expression of K-Ras downstream genes (i.e., CTNNB1, CCND1) was diminished in the treated Kras-positive cells. In conclusion, our combinational platform signifies a new potential for blockade of oncogenic K-Ras and thereby prevention of tumor progression and metastasis. However, further validations are still required regarding the in vitro and in vivo efficacy and safety of this approach.