The c.29T>C polymorphism of the transforming growth factor beta-1 (TGFB1) gene, bone mineral density and the occurrence of low-energy fractures in patients with inflammatory bowel disease.

Gastrointestinal tract conditions are frequently associated with low bone mineral density and increased risk of fractures due to osteoporosis, the latter concerning particularly inflammatory bowel disease (IBD) patients. One of the candidate genes involved in osteoporosis is the transforming growth factor beta-1 (TGFB1) whose polymorphisms may be responsible for the development of this disease. The aim of this study was to analyse the frequency of TGFB1 polymorphic variants and determine the association between the c.29T>C TGFB1 polymorphism, and bone mineral density and fractures in IBD patients. The study subjects included 198 IBD patients [100 suffering from Crohn's disease (CD) and 98 from ulcerative colitis (UC)] and 41 healthy volunteers as a control group. Densitometric bone measurements were obtained using dual energy X-ray absorptiometry. The TGFB1 genotyping was conducted using restriction fragments length polymorphism. We conducted an analysis of genotype distribution's concordance with Hardy-Weinberg equilibrium. We found statistically significant differences in lumbar spine (L2-L4) and femoral neck BMD and T-scores between CD, UC and control subgroups. The distribution of TGFB1 polymorphic variants among CD and UC patients was concordant with Hardy-Weinberg equilibrium. There were no statistically significant differences in densitometric parameters (lumbar spine and femoral neck BMD, T-score, and Z-score) between carriers of different TGFB1 polymorphisms among IBD (CD and UC) patients nor among controls. We have found no statistically significant differences in the prevalence of low-energy fractures between groups of different TGFB1 polymorphic variant carriers. The allele dose effect, recessive effect and dominant effect analysis did not show an association between low-energy fractures and the TGFB1 polymorphisms among CD and UC patients. We have not observed an association between the c.29T>C TGFB1 polymorphic variant and the bone mineral density within the cancellous and cortical bones (L2-L4 and femoral neck, respectively), or the occurrence of fractures among the IBD patients and their family members.

Polymorphisms and allele frequencies of glutathione S-transferases A1 and P1 genes in the Polish population.

Glutathione S-transferases (GST) A1 and P1 are crucial enzymes involved in the biotransformation of drugs, carcinogens, and toxins, and their activity may influence drug response, susceptibility to diseases, and carcinogenesis. The genes encoding these enzymes, GSTA1 and GSTP1, have been examined in many studies because of their genetic variability, which may affect enzymatic activity. The goal of this study was to determine the distribution of the alleles GSTA1*A/*B and GSTP1*A, *B, and *C in the Polish population. A total of 160 subjects from the Polish population were genotyped for 2 polymorphisms (I105V and A114V) in the GSTP1 gene using pyrosequencing. The promoter region of the GSTA1 gene was screened using sequencing. The detected variants were subjected to haplotype analysis. We found that the distribution of the alleles GSTA1*A/*B and GSTP1*A, *B, and *C in the Polish population correspond to the results of studies in Caucasians. Furthermore, we identified additional single nucleotide polymorphisms, excluding 3 well-known changes (G-52A, C-69T, T-567G), which are linked to alleles GSTA1*A/*B, that affect enzyme activity. A total of 4 haplotypes were identified in 160 Polish individuals.

Complete mitochondrial genome of wild aurochs (Bos primigenius) reconstructed from ancient DNA.

Extinct aurochs (Bos primigenius), accepted as the ancestor of domestic cattle, was one of the largest wild animals inhabiting Europe, Asia and North Africa. The gradual process of aurochs extinction finished in Poland in 1627, were the last recorded aurochs, a female, died. Some aspects of cattle domestication history and the distribution of aurochs genetic material among modern cattle breeds still remain unclear. Analyses of ancient DNA (aDNA) from bone sample deliver new genetic information about extinct wild aurochs as well as modern cattle phylogeny. DNA was extracted from a fragment of aurochs fossil bone found in the Pisz Forest, Poland. The sample was radiocarbon-dated to about 1500 yBP. The aDNA was used for Whole Genome Amplification in order to form a DNA bank. Auroch mitochondrial DNA sequences were amplified using sets of 41 primers overlapping the whole mtDNA, cloned and sequenced. The sequence of the whole mitochondrial genome was reconstructed and deposed in GenBank [GenBank:JQ437479]. Based on the phylogenetic analyses of the Bovine mitochondrial genomes, a phylogenetic tree was created. As expected, the tree clearly shows that the mtDNA sequence of the analyzed PWA (Polish Wild Aurochs) individual belongs to haplogroup P. In the course of the comparative mtDNA analysis we identified 30 nucleotide marker positions for haplogroup P and nine unique PWA differences compared to the two remaining haplotype P representatives. Our analysis provides the next step to the reconstruction of the demographic history of this extinct but still exciting species.

Self-incompatibility alleles in Polish wild pear (Pyrus pyraster (L.) Burgsd.): a preliminary analysis.

Wild pear (Pyrus pyraster, syn. P. communis var. pyraster) is thought to be one of the species that gave rise to all other members of the genus Pyrus, although intraspecific hybridizations with cultivated varieties could cause the disappearance of original species characteristics. S-RNase alleles from 7 different wild pear individuals, collected from various regions of Poland, were cloned on the basis of the PCR method and nucleotide sequence analyses. The hypervariable (HV) region is responsible for allele-specific S-RNase activity in the self-incompatibility mechanism. The high level of polymorphism of its sequences may constitute a source of valuable phylogenetic information. From all individuals, 14 sequences were obtained successfully, and 9 of them were novel alleles. Phylogenetic analysis of these alleles was based on the amino acid sequence interpretation of coding regions and intron nucleotide sequences. The research conducted on a limited pool of available P. pyraster alleles gives only an initial insight into possible S-RNase allele polymorphisms in wild populations. At this stage, the results do not confirm a strong influence of cultivated pear species on the wild pear.

Production of ZFN-mediated GGTA1 knock-out pigs by microinjection of gene constructs into pronuclei of zygotes.

Animals as a source of organs and tissues for xenotransplantation could become a backup solution for the growing shortage of human donors. The presence of human xenoreactive anti- bodies directed against Galα1,3Gal antigens on the cell surface of a pig donor triggers the activa- tion of the complement leading to a hyperacute reaction. The development of genetic engineer- ing techniques has enabled the modification of genomes by knocking in and/or knocking out genes. In this paper, we report the generation of modified pigs with ZFN mediated disruption of the GGTA1 gene encoding the enzyme responsible for synthesis of Galα1,3Gal antigens. ZFN plasmids designed to target the exon 9 region of the pig GGTA1 gene encoding the catalytic domain were injected into the pronuclei of fertilized egg cells. Among 107 piglets of the F0 gene- ration analyzed, one female with 9-nt deletion in exon 9 of the GGTA1 gene was found. 13 of 33 piglets of the F1 generation represented the +/- GGTA1 genotype and 2 of 13 F2 piglets repre- sented the -/- GGTA1 genotype. No changes in the animals' behavior, phenotype or karyotype were observed. Analysis confirmed heredity of the trait in all animals. A complex functional analysis of the modified animals, including flow cytometry, human serum cytotoxicity test and immunohistochemical detection, was performed to estimate the phenotype effect of genetic modification and this indicated an efficient GGTA1 knock-out in modified pigs.

Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle.

This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test was correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes. The expression of Bcl-2 and Bax genes was estimated on the mRNA and protein levels, respectively using real-time PCR and Western-blotting. To evaluate developmental competence of these oocytes, cumulus-oocyte complexes classified as BCB+ (low activity of G6PDH), BCB- (high activity of G6PDH) and Control were used for in vitro embryo production. In immature oocytes, the Bax transcript level in BCB- oocytes was significantly higher (P<0.001) in comparison to Control. In mature oocytes, the Bcl-2 transcript level was significantly lower in BCB+ oocytes (P<0.01) and in BCB- oocytes (P<0.05) in comparison to Control. However, no relation was found between the activity of G6PDH and the expression of the Bcl-2 or Bax proteins, both in immature and mature oocytes. Our results on the transcript level seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. However, results obtained at the protein level did not confirm this conclusion. The usefulness of the BCB test as the indirect marker of apoptosis seems to be questionable. The lack of significant differences in the blastocyst rates developed from BCB+ and Control oocytes decreases the validity of BCB test in IVP technology.

Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection.

The aim of our study was to determine the in vitro developmental potential of porcine nuclear-transferred (NT) embryos that had been reconstructed with Tg(pWAPhGH-GFPBsd) transgene-expressing fibroblast cells. The gene construct was introduced into fibroblast cells by the novel method of nucleofection or standard lipofection. NT oocytes derived from foetal and adult dermal fibroblast cells were stimulated by either simultaneous fusion and electrical activation (Groups IA and IB) or sequential electrical and chemical activation (Groups IIA and IIB). The percentages of cloned embryos that reached the morula and blastocyst stages were 152/254 (59.8%) and 77/254 (30.3%) or 139/276 (50.4%) and 45/276 (16.3%) in Groups IA or IB, respectively. The rates of NT embryos that developed to the morula and blastocyst stages were 103/179 (57.5%) and 41/179 (22.9%) or 84/193 (43.5%) and 27/193 (14.0%) in Groups IIA and IIB, respectively. In conclusion, the in vitro developmental competences of porcine transgenic NT embryos that had been reconstructed with the Tg(pWAPhGH-GFPBsd) gene-transfected fibroblast cells were relatively high. Further, the nucleofection efficiency of all the porcine fibroblast cell lines as estimated by intra-vitam fluorescent evaluation based on the index of reporter eGFP transgene expression was nearly 100%. However, PCR analysis for transgene screening confirmed the absence of Tg(pWAPhGH-GFPBsd) fusion gene in some of the nucleofected cell lines. To our knowledge, the novel method of nucleofection is the first to transfect nuclear donor cells in the production of transgenic cloned embryos.

Bone mineral density and the 570A>T polymorphism of the bone morphogenetic protein 2 (BMP2) gene in patients with inflammatory bowel disease: a cross-sectional study.

Finding genetic predictors of osteoporosis and fractures in patients with inflammatory bowel disease (IBD) may provide incentives for non-pharmacological actions and so improve the long-term prognosis of the patients. We analysed the incidence of BMP2 570A>T polymorphic variants and their association with bone mineral density (BMD) and the incidence of fractures in patients with IBD. The study comprised 198 IBD patients (100 with Crohn's disease (CD), and 98 with ulcerative colitis, (UC)) and 41 healthy controls. Bone densitometric analysis was carried out using the DXA method. The 570A>T polymorphisms in the BMP2 gene were genotyped using RFLP. We found significant differences in the BMD and T-scores of the lumbar spine (L2-L4) and femoral neck between the three groups. In controls and CD patients, the highest L2-L4 BMD was found in carriers of the AA variant of the BMP2 gene, while among UC patients it was the case of TT carriers. In both femoral neck and lumbar spine among UC patients, the highest BMD was observed in carriers of the TT variant of the BMP2 gene. Among patients with CD and in the control group, the highest L2-L4 BMD was found in carriers of the AA variant, whereas in UC patients, it was the case of TT homozygotes. Within the femoral neck, there were no significant differences in BMD for the carriers of individual variants of BMP2 gene polymorphism. We conclude that the 570A>T polymorphism of the BMP2 gene, no statistically significant relationship was observed between the polymorphic variant and bone mineral density or the incidence of fractures in IBD patients.

Estimation of the number of genes specifying the heavy chain of mouse thymus leukemia antigens.

We have investigated the number of structural genes present in ASL-1 cells, a murine leukemia cell line which encodes the heavy chain of thymus leukemia (TL) antigens, a protein which is similar to the Class I histocompatibility antigens. TL-specific messenger RNA was purified from polysomes of ASL-1 cells by immunoprecipitation, and this messenger RNA translated in vitro to produce a Mr 42,000 protein which comigrated on acrylamide gel with nonglycosylated TL heavy chain. A 32P-labeled complementary DNA (cDNA) was synthesized by reverse transcription of the TL-specific messenger RNA as template. Analysis of reassociation kinetics of the 32P-TL-cDNA with DNA from ASL-1 cells showed that the kinetics was indistinguishable from that obtained using a DNA encoding a single-copy gene (C mu). An analysis was performed in which DNA from ASL-1 cells was subjected to digestion with each of three restriction enzymes and hybridized with 32P-TL-cDNA according to the Southern "blot" technique. Two bands formed hybrids with the 32P-TL-cDNA with each of three restriction enzymes used. These data are consistent with the presence of a small number of structural genes for the TL heavy chain in the genome of ASL-1 leukemia cells.

Two coexisting heterozygous frameshift mutations in PROP1 are responsible for a different phenotype of combined pituitary hormone deficiency.

The role of genetic background in childhood-onset combined pituitary hormone deficiency (CPHD) has been extensively studied. The major contributors are the PROP1, POU1F1, LHX3, LHX4 and HESX1 genes coding transcription factors implicated in pituitary organogenesis. The clinical consequences of mutations encompass impaired synthesis of a growth hormone (GH) and one or more concurrent pituitary hormones (i.e. LH, FSH, TSH, PRL). Manifestation of the disorder may vary due to various mutation impacts on the final gene products or an influence of environmental factors during pituitary organogenesis. We describe the clinical and molecular characteristics of two brothers aged 47 and 39 years presenting an uncommon manifestation of congenital hypopituitarism. Sequencing of the PROP1, POU1F1, LHX3, LHX4 and HESX1 genes was performed to confirm the genetic origin of the disorder. A compound heterozygosity in the PROP1 gene has been identified for both probands. The first change represents a mutational hot spot (c.150delA, p.R53fsX164), whereas the second is a novel alteration (p.R112X) that leads to protein disruption. Based on precise genetic diagnosis, an in silico prediction of a p.R112X mutation on protein architecture was performed. The resulting clinical phenotype was surprisingly distinct compared to most patients with genetic alterations in PROP1 reported in the current literature. This may be caused by a residual activity of a newly identified p.R112X protein that preserves over 70 % of the homeodomain structure. This examination may confirm a key role of a DNA-binding homeodomain in maintaining PROP1 functionality and suggests a conceivable explanation of an unusual phenotype.

Immunity to murine leukemia induced in susceptible mice by transfected mouse fibroblasts.

A cultured cell line of mouse fibroblasts was transfected with DNA from murine leukemia cells expressing a previously characterized tumor-associated antigen. Antigen-positive cells were used as immunogens in an immunotherapy protocol to determine if they stimulated resistance to the malignant proliferation of the leukemia in susceptible mice. For the experiments, LM(TK-) mouse fibroblasts, a thymidine kinase-deficient mouse cell line, were cotransfected with DNA from ASL-1 murine leukemia cells and the plasmid pSV2neo conferring resistance to Geneticin. Integration of the plasmid into cellular DNA was confirmed by restriction digest blot analysis. A/J mice, highly susceptible to the malignant proliferation of passively transferred ASL-1 leukemia cells, were immunized with the transfected cells. Animals receiving two prior injections of antigen-positive transfected cells and then challenged with an injection of viable ASL-1 cells survived longer than animals in the unprotected control group or in the group receiving immunizations with LM(TK-) cells transfected with plasmid only (p less than 0.01). Some of the mice appeared to have rejected the tumor and lived more than 80 days. One group of protected animals rechallenged with a second injection of ASL-1 cells, 40 days after the first, survived for more than 50 additional days, without evidence of recurrent disease.

The current state of xenotransplantation.

Pigs as a source of grafts for xenotransplantation can help to overcome the rapidly growing shortage of human donors. However, in the case of pig-to-human transplantation, the antibody-xenoantigen complexes lead to the complement activation and immediate hyperacute rejection. Methods eliminating hyperacute rejection (HAR) include α1,3-galactosyltransferase (GGTA1) inactivation, regulation of the complement system and modification of the oligosaccharide structure of surface proteins. The humoral immune response control and reduction of the risk of coagulation disorders are the priority tasks in attempts to overcome acute humoral xenograft rejection that may occur after the elimination of HAR. The primary targets for research are connected with the identification of obstacles and development of strategies to tackle them. Because of the magnitude of factors involved in the immune, genetic engineers face a serious problem of producing multitransgenic animals in the shortest possible time.

Yellow lupine cyclophilin transcripts are highly accumulated in the nodule meristem zone.

Cyclophilin (CyP) is one of the enzymes that act as peptidylprolyl cis-trans isomerases (EC 5.2.1.8). The cDNA and an intronless gene coding for cytosolic CyP have been isolated from yellow lupine. The deduced amino acid sequence of the characterized open reading frame shows approximately 80% homology with cytosolic CyP from other organisms. Southern blots of genomic DNA indicate that there is a small family of genes for CyP-related genes in the yellow lupine genome. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs. The amount of CyP transcripts is dramatically increased in root nodules. In situ hybridization experiments indicate that CyP transcripts are localized mainly in meristematic tissues, with the highest level observed in the nodule meristem zone. The promoter of the sequenced gene contains 5' AAAGAT 3' and AT-rich motifs that are characteristic for some nodulin promoters.

Deletion screening and carrier detection in Duchenne muscular dystrophy in Polish population via direct analysis of DNA and RNA transcripts.

Analysis of 102 Polish Duchenne/Becker muscular dystrophy (D/BMD) patients was performed by 'multiplex' amplification of 22 fragments of the DMD/BMD gene and deletions were found in 55% of the patients. The data obtained using PCR were compared with results of 25 Southern blotting and hybridization experiments with cDNA probes and with immunostaining using anti-dystrophin antibodies. In order to determine more precise deletion breakpoints, additional experiments were performed on dystrophin transcripts isolated from peripheral blood lymphocytes. These data found direct application in carrier analysis in the respective families by detection or exclusion of aberrant cDNA fragments. Carrier detection was also performed by RFLP-PCR, analysis of polymorphic (CA)n repeats and single stranded conformational polymorphism (SSCP) for selected exons of the DMD gene.

Oligonucleotide DNA fingerprinting: results of a multi-center study on reliability and validity.

We report the results of an empirical study of 256 paternity cases referred to 7 different German laboratories for DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5. All parameters characteristic of such multilocus DNA fingerprints were found to differ significantly between the contributing centres. Despite these differences, clear-cut decisions between paternity and non-paternity could be made in all but one case. Furthermore, we found no systematic deviation of the gel-phenotype distribution among trios from random expectation as derived from commonly adopted analytical models. Thus, we conclude that oligonucleotide DNA fingerprinting is a robust and reliable means for the resolution of paternity cases.

Some aspects of structure of murine H-2d antigens.

The apparent molecular weight of proteins precipitated by normal rabbit serum from lysates of lymph node cells ranged from 10 000 to 150 000. Mono-specific anti-H-2d serum precipitated molecules composed of heavy and light chains with Mr of 47 000 and 12 000, respectively. Tunicamycin treatment caused a decrease of the molecular weight of heavy chain by 4 000 and a change of pI from 5.2 - 5.9 to 5.4 - 5.8. Non-glycosylated heavy chain exhibited Mr of 43 000. The oligosaccharide side chains were resistant to digestion by endo-beta-N-acetylglucosaminidase H. The Mr and pI of beta 2-microglobulin were not altered by treatment with tunicamycin and endo-beta-N-acetylglucosaminidase H. Tunicamycin caused a 50% decrease of overall synthesis of cellular proteins.

Application of non-radioactive methods of DNA detection in analysis of human genetic disorders.

We have applied two non-radioactive methods for detection of unique sequences in human genome: 1 polymerase chain reaction, 2 hybridization with digoxigenin-deoxyuridine 5-triphosphate labeled probes. With the polymerase chain reaction technique we were able to amplify short segments of genes coding for coagulation factors VIII and IX. Electrophoretical analysis of products of polymerase chain reaction enabled us to detect deletions causing hemophilia A or B. To analyse deletions in dystrophin gene, the most frequent cause of Duchenne muscular dystrophy, we have amplified several different fragments of this gene simultaneously. We have studied restriction fragment length polymorphism closely linked to the cystic fibrosis locus with digoxigenin-deoxyuridine 5 -triphosphate labeled probe p3.11 with sensitivity comparable to methods involving the use of radioisotopes.

Partial purification, characterization and cell-free translation of mRNAs coding for H-2d antigens.

Microsomal RNA from SL2 cells was fractionated on oligo(dT)cellulose into three fractions: I was very similar to the non-fractionated RNA, II contained 28S and 18S rRNA, III [poly(A)RNA] of 4 - 50S, constituted 1.0-2.1% of the total RNA pool and was still contaminated with rRNAs. The last fraction was synthesized at the highest rate. Anti-H-2d serum precipitated cell-free translation products of 17S (heavy chain) and 10S (beta 2-microglobulin) mRNA fractions obtained by centrifugation on 10-30% linear sucrose gradient. beta 2-Microglobulin synthesis was 5-fold higher as compared with the heavy chain synthesis. Anti-H-2d serum precipitated translation products with Mr of 47 000-48 000 (H-2Dd and H-2Kd heavy chains) and 12 000 (beta 2-microglobulin). With liver mRNA, the patterns of translation products were identical, but beta 2-microglobulin synthesis was lower by a half than heavy chain synthesis. H-2d heavy chain messenger constituted 0.3-0.4% of the enriched fraction and was purified 15-20--fold.

Haplotype analysis of phenylalanine hydroxylase alleles in polish families with phenylketonuria.

Eight polymorphic restriction enzyme sites at phenylalanine hydroxylase locus from the parental chromosomes in Polish families with phenylketonuria were analyzed. Among 28 chromosomes studied, we identified haplotypes found within the Danish population. Haplotype 2 has been found in 25% of affected alleles. One of the patients studied is homozygous for this haplotype.

Paternity testing with oligonucleotide multilocus probe (CAC)5/(GTG)5: a multicenter study.

The statistical analysis is reported of 256 paternity cases referred to seven different German laboratories for multilocus DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5 and restriction enzyme HinfI. All parameters characteristic of multilocus DNA fingerprints were found to differ significantly between the contributing centres: the number of analyzed gel positions, the number of bands scored per individual, the probability of occurrence of a band at a particular position, and the band-sharing probabilities between the mother and both child and alleged father. Despite these differences, paternity cases could be divided clearly into two distinct subgroups on the basis of (i) offspring bands that could not be assigned to either the mother or the alleged father and (ii) the extent of band-sharing between child and alleged father. This partitioning, which is likely to correspond to true and false paternity, confirms previous findings for other multilocus probes. A goodness-of-fit test on the normalized number of bands scored per individual revealed no systematic deviations from commonly adopted analytical models regarding electrophoretic bands as independent entities. Log10-likelihood ratios of paternity vs. non-paternity were calculated utilizing one of these models, and a clear-cut partitioning was again obtained which coincides with that mentioned before. Only one case could not be decided unambiguously, and was either due to two independent mutations or to a close relative of the alleged father being the true father.