Tadeusz Malewski

COMPUTER ANALYSIS OF DISTRIBUTION OF PUTATIVE CIS- AND TRANS-REGULATORY ELEMENTS IN MILK PROTEIN GENE PROMOTERS

Multiple alignment of 28 milk protein gene promoters belonging to seven protein superfamilies is described. In these gene promoters three groups of common motifs were found: group I--specific for all milk protein gene promoters; group II--specific only for one gene superfamily; and group III--motifs shared by several gene superfamilies. Motifs of group I and III do not have any preferential location in the promoters, while group II motifs are located in the proximal part, from -36 to -224. Milk protein gene promoters were analysed for presence of putative binding sites for nine transcription factors important for the expression of this group of genes. The transcription factor binding sites for C/EBP, CTF/NF1, MAF and MGF were found in all promoters investigated. The set of putative transcription factor binding sites or response elements for GRE, IRE, PMF, STR and YY1 is unique for every gene superfamily.

Single nucleotide polymorphism in the promoter region of the lactoferrin gene and its associations with milk performance traits in Polish Holstein-Friesian cows

Bovine lactoferrin (LTF) is a multifunctional small glycoprotein found in milk acting mainly as a defense factor in the mammary gland. Many polymorphisms have been found in the bovine LTF gene but almost none were considered as genetic markers of production traits in dairy cattle. In this study, the promoter fragment of LTF gene containing mutation (G/C) in position +32 has been amplified by PCR followed by genotyping by the SSCP and RFLP method. Three hundred fifty-eight Polish Holstein-Friesian cows were screened, giving the following frequency of genotypes: 0.628, 0.313 and 0.059 for GG, GC and CC, respectively. GLM (General Linear Model) analysis was applied to evaluate the associations of lactoferrin with milk performance traits, including SCC (somatic cell count). It was found that CC cows show significantly higher (P ≤ 0.01) protein content in milk in comparison with GG cows. The values of other milk performance traits were also higher but at nonsignificant levels. SCC in milk was the lowest in CC cows, but also at a nonsignificant level.

Changes in the GnRH mRNA and GnRH receptor (GnRH-R) mRNA levels in the hypothalamic-anterior pituitary unit of anestrous ewes after infusion of GnRH into the third cerebral ventricle.

In the present paper the role of GnRH in the ultrashort loop of the negative feedback action on GnRH secretion was evaluated on the molecular level by the Real-time PCR technique. Specifically, the effect of GnRH infused into the third cerebral ventricle on the expression of GnRH and GnRH receptor (GnRH-R) genes was analyzed in the hypothalamic-pituitary unit of anestrous ewes. GnRH did not significantly affect GnRH mRNA levels in the preoptic/anterior hypothalamic area but drastically increased its level in the ventromedial hypothalamus. In addition, GnRH infusion augmented GnRH-R mRNA level in the entire hypothalamus. In the GnRH-treated animals, anterior pituitary GnRH-R mRNA level and plasma LH concentration were also elevated. The changes in GnRH mRNA and GnRH-R mRNA levels in the hypothalamus in response to treatment with GnRH suggest that GnRH acts differently on the stability of these transcripts. On the basis of presented results it seems that GnRH may affect GnRH and GnRH-R biosynthesis and, consequently, GnRH/LH release.

Transcription factor binding to variable nucleotide sequences in 5′-flanking regions of bovine casein genes

Different transcription-factor (TF)-binding sites involved in expression of bovine milk protein genes were identified in their 5′-flanking regions (promoters). Previous studies have identified polymorphism within 5′-flanking regions of bovine milk protein genes resulting from the natural variations in nucleotide sequences. Our computer analysis using the TESS program revealed that in the bovine casein genes at least some of the polymorphic sequences are located in putative TF-binding sites, and thus they may influence gene expression. Moreover, in the 5′-flanking region of the bovine αS1-casein gene two putative mammary gland factor/signal transducer and activator of transcription binding sites were identified differing in the G/A nucleotide substitution. Electrophoretic mobility-shift assays showed quantitative differences in the binding of nuclear proteins to variable sequences in bovine αS1- and αS2-casein gene promoters. The differences in protein binding to variable sites of bovine αS2-casein gene at positions –1101/−1100 and −186 were statistically significant (p<0.05). Results of electrophoretic mobility-shift assays competition experiments suggested that TFs such as activator protein 1, cAMP-response element-binding protein, thyroid receptor and Ying Yang 1 might take part in the formation of DNA–protein complexes. Using reverse transcription PCR method, differences in the transcription rates were found for the αS2-casein gene promoter variants, but not in the case of αS1 gene variants.

Towards an integrated approach to study SNPs and expression of candidate genes associated with milk protein biosynthesis

MilkProtChip is oligonucleotide microarray allowing bovine genotyping based on single nucleotide polymorphisms (SNPs) in genes influencing milk protein biosynthesis. A total of 71 SNPs in 42 genes were selected as associated with milk protein biosynthesis. Genotyping of about 300 animals of Polish Black-and-White cattle showed that SNPs in acyl-CoA:1,2-diacylglycerol O-transferase (DGAT1), lactoferrin (LTF), casein kappa (CSN3) and growth hormone receptor (GHR) genes were associated with several milk performance traits. Analysis of correlations between SNPs and milk production traits showed that SNPs in single genes rarely affect the investigated traits. Only 4 of 42 investigated single SNPs had impact on milk production traits while 22 combinations of paired SNPs in these genes had impact. Positive effect SNP combinations in two genes can be a result of additive effect on these SNPs on the same traits or effect of genes interaction. The MilkBovExp chip representing 90 genes encoding transcription factors expressed in the bovine mammary gland and/or involved in mammary gland signaling pathways was designed for further investigation of impact of gene expression and/or its encoded products on milk traits performance.

Expression Profiling of Candidate Genes for Abdominal Fat Mass in Domestic Chicken Gallus gallus

The quantitative traits of mass and percentage of abdominal fat in chicken and various types of obesity in mammals are homologous and functionally similar. Therefore, the genes involved in obesity development in humans and laboratory rodents as well as those responsible for pig lard thickness could be involved in abdominal fat deposition in broilers. Expression of candidate genes FABP1, FABP2, FABP3, HMGA1, MC4R, PPARG, PPARGC1A, POMC and PTPN1 was studied in fat, liver, colon, muscle, pituitary gland, and brain in chicken (broilers) using real-time PCR. Significant difference in the HMGA1 gene expression in the liver of broiler chicken with high (3.5 ± 0.18%) and low (1.9 ± 0.56%) abdominal fat concentration has been revealed. The expression of this gene was been shown to correlate with the amount (0.7, P ≤ 0.01) and mass (0.7, P ≤ 0.01) of abdominal fat. The PPARG gene expression in liver in the same chicken subsets was also significantly different. Correlation coefficients of the gene expression with the abdominal fat amount and mass were respectively 0.55 (P ≤ 0.05) and 0.57 (P ≤ 0.01). Based on these results, we suggest that the HMGA1 and PPARG genes are involved in abdominal fat deposition. The search for single nucleotide polymorphisms (SNPs) in the HMGA and PPARG regulatory regions could facilitate identifying genetic markers for broiler breeding according to the mass and percentage of abdominal fat.

MORPHOLOGICAL AND MOLECULAR FEATURES OF PUNCTODERA STONEI BRZESKI, 1998 (NEMATODA: HETERODERIDAE) - SPECIES ASSOCIATED WITH ROOTS OF GRASSES

Abstract. This article provides morphological and molecular characteristics of Punctodera stonei Brzeski, 1998. Comparison of partial sequences of 18S and 28S rDNA genes from P. stonei sampled in Poland and Punctodera sp. from Canada showed their 100% similarity. This is the first report on the occurrence of P. stonei outside of Europe. We provide data on morphology of males and 2nd stage juveniles of this species and an identification key to males of the genus Punctodera Mulvey et Stone, 1976. Moreover, the paper presents evolutionary relationships of P. stonei within the family Heteroderidae.

Involvement of Fas/FasL pathway in the murine model of atopic dermatitis

Objective and designThe aim of this study was to elucidate the role of apoptosis mediated through Fas/FasL pathway using the mouse model of atopic dermatitis (AD).Materials and treatment AD was induced by epicutaneous application of ovalbumin (OVA) in wild-type C57BL/6, B6. MRL-Faslpr/J (Fas−) and B6Smn.C3-Faslgld/J (FasL−) mouse strains.MethodsSkin samples were subjected to staining for Fas/FasL expression, M30 epitope and assessment of inflammatory response via immunohistochemical staining. Cytokine and chemokine production was assessed by real-time PCR.ResultsIn comparison to wild-type mice, OVA sensitization of Fas- and FasL-deficient mice led to increased epidermal and dermal thickness, collagen deposition and local inflammation consisting of macrophages, neutrophils and CD4+ T cells. Fas- and FasL-deficient mice showed increased total counts of regulatory T cells (Tregs) and IgE levels in blood as well as increased expression of IL-1β, IL-4, IL-5, IL-13 and TGF-1β mRNA in comparison to wild-type mice. On the other hand, expression of CXCL9 and CXCL10, IL-17 mRNAs in the skin samples in Fas- and FasL-deficient mice was decreased.ConclusionsOur results show that lack of the Fas-induced apoptosis leads to exacerbation of AD characteristics such as Th2 inflammation and dermal thickening. Therefore, Fas receptor can play an important role in AD pathogenesis by controlling development of the local inflammation.

Design of a system for genotyping of Gallus gallus based on the rSNP (Regulatory single nucleotide polymorphism) alleles affecting the egg shell thickness

PCR amplification of the six fragments of regulatory and coding regions of chicken ChEST985k21 gene (accession no. CR523443), substantially affecting the egg shell thickness quantitative trait, was carried out. Sequencing of these fragments in six chickens from a native Polish breed, Green-legged Partridgenous, with different manifestation of the trait of interest enabled identification of six single nucleotide polymorphism (SNP) sites within the ChEST985k21 sequence. Five of these sites were located in the regulatory region, and one site, in the coding region. For all SNPs identified, the existence of transcription factor binding sites, present in only one allelic variant, was demonstrated. This finding enables considering these sites as regulatory single nucleotide polymorphisms, rSNP. The effect of rSNP discovered on the chicken egg shell thickness was tested using PCR amplification with allele-specific primers. In the groups of chicken of Rhode Island Red breed with thick (389.9 ± 13.09 μm) and thin (315.7 ± 21.38 μm) egg shells statistically significant differences in the allele frequencies of the ST2_1, ST3_1, ST3_2, and ST3_3 polymorphic sites. In the same groups of birds, statistically significant differences in the shell thickness were observed in the rSNP allele genotypic classes ST2_1, ST3_1, ST3_2, ST3_3, and ST6_1. Based on these data, it was concluded that rSNPs influenced manifestation of the quantitative trait examined, and the genotyping system for marker assisted selection was constructed.

Alternative Transcription of Peroxisome Proliferator-Activated Receptor Gamma in the Liver Is Associated with Fatness of Chickens

The expression of four transcription variant of peroxisome proliferatoractivated receptor gamma gene (PPARG) (XM_015292931.1; XM_015292932.1; XM_015292933.1 and NM_001001460.1) in the liver of broilers was measured and its correlation with abdominal fat weight and relative abdominal fat content was investigated. The study was conducted with 92 slow-growing crossbred chickens (Cobb males x indigenous Green-legged Partridge female chickens) divided into “fat” and “lean” groups, according to their abdominal fat yield. The NM_001001460.1 transcriptwas upregulated with ratio of means 4.26 (p≤0.01) in the “fat” group in relation to the “lean” group. Expression of this transcript was highly correlated with relative abdominal fat content (0.71, p≤0.01) and abdominal fat weight (0.59, p≤0.01). Two SNPs are located in putative transcription factor binding sites. Mutation -991C>A disrupts PPAR while mutation -884C>T disrupts C/EBP putative binding site. The gene expression analysis of PPARg showed that the expression of the transcripts (NM_001001460.1) was more than four times higher in fat than in lean chickens. These results point out that the peroxisome proliferator-activated receptor gamma NM_001001460.1 transcript could be candidate gene for determination of abdominal fat deposition in the chickens.