Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette

A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus. The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q). Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.     

Increase of probability of cell loss in the endothelium of blood vessel capillaries in progeny of rats after a low-dose irradiation of one of the parents (electron microscopy study)

The total gamma-irradiation of Wistar rats at a dose of 0.25 Gy as well as at higher doses (0.5, 2 and 4.5 Gy) produces in the capillary endothelial cells of myocardium and lung a pronounced, dose-independent increase of the yield of necrotized cells. Similar changes were revealed in the animals, of which one of the parents (a male one day, a female seven days prior to copulation) was irradiated at doses of 0.25 and 0.5 Gy. This effect was observed in all studied descendants. The massive induction of the changes already by low radiation actions and their dose-independence allow considering the revealed effects as a manifestation of peculiar cellular reactions that presumably have epigenetic nature.     

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.     

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.     

Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli

We have used GET Recombination, an inducible homologous recombination system for Escherichia coli, to insert one of the most common thalassaemia mutations into the intact β-globin locus in a second generation BAC vector. We first inserted a PCR fragment carrying the tetracycline resistance gene (TetR) into the β-globin gene. All recombinant clones examined contained the TetR gene at the correct target site. Next, a PCR fragment with the IVS I-110 G→A splicing mutation but no selectable marker was used to replace the TetR gene in a second round of GET Recombination. Recombinant clones were selected by plating on medium containing chlorotetracycline and fusaric acid. Although counterselection for the TetR gene is not very efficient, four recombinant colonies with the IVS I-110 mutation were identified among 480 clones screened. Analysis of the recombinant clones did not show any other modifications or rearrangements. Thus the TetR gene can be used in combination with GET Recombination to introduce point mutations and other modifications in BACs without leaving behind any operational sequences, in order to generate accurate cell and transgenic mouse models for various diseases.     

A study of the prevalence of regulatory genes controlling virulence gene expression among Vibrio choleraeeltor biovariant strains varying in their pandemic potential

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.     

Luminophores of the luminous fungus <Emphasis Type="Italic">Neonothopanus nambi</Emphasis>

Isolation of luminophores from the mycelium of a luminous fungus Neonothopanus nambi is reported. In addition to the emission peak with a maximum at 520–530 nm (the wavelength of visible green light) that corresponded to the maximum of light emission by the fungus in vivo, the fluorescence spectra of the raw extracts contained a peak with a maximum in the visible blue-light range. The luminophore that emitted the blue light was an individual compound with a molecular weight of 894 Da. Calculations that took the isotope composition of chemical elements into account pointed at C₅₂H₆₅N₂O₁₁, C₅₁H₆₅N₄O₁₀, C₅₃H₆₁N₆O₇, C₄₇H₆₅N₄O₁₃, and C₄₆H₆₅N₆O₁₂ as the putative chemical formulae of the luminophore. A sample that contained substances of a yellow color was obtained; these substances emitted fluorescence at the wavelengths of green visible light. The luminophores in this sample probably included riboflavin or derivatives thereof (flavin mononucleotide or flavin adenine dinucleotide).     

Pyruvate kinase in the bovine adrenal cortex

Pyruvate kinase from bovine adrenal cortex was purified to an electrophoretically homogeneous state. The molecular weight of the native enzyme is about 230 000, that of one subunit is 57 000. The maximal values of the pyruvate kinase initial reaction rate were obtained in 50 mM imidazole-acetate buffer within the pH range of 6.8 to 7.0. The curve of the initial pyruvate kinase reaction rate versus phosphoenolpyruvate (PEP) and ADP concentrations is hyperbolic and obeys the Michaelis-Menten kinetics with Km for PEP and ADP of 0.055 X 10(-3) M and 0.25 X 10(-3) M, respectively. The enzyme is activated by Mn2+ and Co2+ by 43 and 38%, respectively. IDP, GDP, and UDP may be used as analogs of ADP. The enzyme is not activated by fructose-1.6-diphosphate and is inhibited by L-phenylalanine and ATP.     

Inheritance and phenotype expression of functional and null alleles of aromatic alcohol dehydrogenase (CAD) in diploid wheats

Functional F and null 0 alleles of the CAD1 (Aadh1) gene, which controls the biosynthesis of aromatic alcohol dehydrogenase, were studied in hybrids of the diploid wheat T. monococcum L. and Triticum sinskajae A. Filat. et Kurk. The gene CAD1 is located in chromosome 5A and is linked with the awnless gene awnS (La) with a recombination frequency of about 32%. Plants with genotypes FF, F0, and 00 were significantly different in the height and mechanical strength of the stalk (culm). The elastic limit of the culm tissues of plants FF was considerably higher than in 00 plants. F0 heterozygotes had intermediate values. The thickness of the wall of the sclerenchyma was thinner in plants with genotype 00. The chemical structure of lignin of plants with the functional CAD allele contained units of a phloroglucinol series missing in the mutant plants. The CAD genotypes had no effect on the relative content of cellulose and lignin in stalks of diploid wheat and insignificantly influenced the ratio of H: G: S units in the lignin structure, as well as some components of extractives. IR-spectroscopy found differences in the distribution of components of cell walls and extractives on the outer and inner surfaces of the culm. The results are discussed in relation to the applied aspects of the use of herbal products. Samples of diploid wheat with various genotypes of CAD can be used as model objects in breeding and genetic research.