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1

Structure, expression and engineering of milk protein genes

100%
Zwierzchowski L.
Biotechnologia
|
1996
|
issue 2
81-94
EN
Expression of milk protein genes is regulated by hormones, growth factors and extracellular matrix.Prolactin, the major lactogenic hormone, promotes all stages of casein gene expression. Besides prolactin, also growth hormone may directly induce expression of milk protein genes. Computer analysis, mutation experiments and DNA-protein binding experiments enabled indentification of mammary-specific trasnscription factors and cis-regulatory sequences in milk protein gene promoters. Also, transgene technology enables recognition of regulatory sequences in milk protein genes.Moreover, transgenic animals carrying structural genes of human proteins fused to mammary specific promoters are considered as living "bioreactors" designated to produce human proteins for pharmaceutical use.
2

Biotechnology of the mammary gland - perspectives of genetic engineering of milk proteins

100%
Zwierzchowski L.
Biotechnologia
|
1998
|
issue 2
33-56
EN
In this article, state of art and perspectives in mammary gland biotechnology are reviewed. Recent progress in recombinant DNA technology as well in embryo manipulation and transfer has made the introduction of specific genes into the germline of animals relatively easy. With appropriate genetic constructs, the expression of the inserted genes in transgenic animals can be controlled in a tissue-specific and in a differentiation-specific manner. Thus, it is now possible to consider alteration of the composition of milk produced by lactating animals in a variety of ways. There is a growing list of foreign milk proteins that have been expressed, and one can envisage placing almost any protein gene of interest under the control of promoter of a milk protein gene. Many human proteins of a potential pharmaceutical use may be now produced in the mammary glands of laboratory or farm animals. Modification of milk composition can be extended not only to produce proteins of commercial value but also, by manipulation of key metabolic enzymes, to fat, lactose, and other components of milk. Many alternations in ruminants' milk composition, including 'humanization' of cow's milk, are planned, however, these manipulations must avait the development of totipotent embryonic cell lines (ESC) of farm animals, cells that enable gene manipulation by homologous recombination. In spite of a great progress, many obstacles and difficulties still exist on the way to economical production of human paharmaceuticals in farm 'transgenic bioreactoractors'. These dificulties are discussed in detail.
3

Polymerase chain reaction-heteroduplex (PCR-HD) polymorphism within the bovine STAT5A gene

64%
Flisikowski K., Zwierzchowski L.
Journal of Applied Genetics
|
2003
|
vol. 44
|
issue 2
185-189
EN
Signal transducers and activators of transcription (STATs) are transcription factors mediating signals of various hormones and cytokines. STAT5A, previously known as the mammary gland factor (MGF), mediates the action of prolactin on milk protein gene expression in mammary epithelial cells. We used polymerase chain reaction-heteroduplex (PCR-HD) and sequencing methods for detection of nucleotide sequence polymorphism in intron 15 of the bovine STAT5A gene. A 281-bp gene fragment, from nt 12525 to nt 12806 (GenBank AJ 237937), was amplified with PCR, denatured and subjected to polyacrylamide gel electrophoresis to detect PCR-HD polymorphism. Three genotypes and two alleles were identified. DNA samples derived from homozygotes AA and BB were sequenced. A trinucleotide CCT deletion was found in the variant B of the STAT5A gene at position 12549. In a group of 72 beef bulls of various breeds and 49 Friesian bulls genotyped by PCR-HD mostly the AA genotype was found (from 83 to 61% depending on breed). The frequency of allele A varied between 0.91 and 0.77. Animals of genotype BB were found in Charolaise and Limousine breeds only.
4

Molecular evolution of coding and non-coding sequences of the growth hormone receptor (GHR) gene in the family Bovidae

64%
Maj A., Zwierzchowski L.
Folia Biologica
|
2006
|
vol. 54
|
issue 1-2
31-36
EN
The GHR gene exon 1A and exon 4 with fragments of its flanking introns were sequenced in twelve Bovidae species and the obtained sequences were aligned and analysed by the ClustalW method. In coding exon 4 only three interspecies differences were found, one of which had an effect on the amino-acid sequence ? leucine 152 proline. The average mutation frequency in non-coding exon 1A was 10.5 per 100 bp, and was 4.6-fold higher than that in coding exon 4 (2.3 per 100 bp). The mutation frequency in intron sequences was similar to that in non-coding exon 1A (8.9 vs 10.5/100 bp). For non-coding exon 1A, the mutation levels were lower within than between the subfamilies Bovinae and Caprinae. Exon 4 was 100% identical within the genera Ovis, Capra, Bison, and Bos and 97.7% identical for Ovis moschatus, Ammotragus lervia and Bovinae species. The identity level of non-coding exon 1A of the GHR gene was 93.8% between species belonging to Bovinae and Caprinae. The average mutation rate was 0.2222/100 bp/MY and 0.0513/100 bp/MY for the Bovidae GHR gene exons 1A and 4, respectively. Thus, the GHR gene is well conserved in the Bovidae family. Also, in this study some novel intraspecies polymorphisms were found for cattle and sheep.
5

Polymorphism within the bovine estrogen receptor-a gene 5?-region

64%
Szreder T., Zwierzchowski L.
Journal of Applied Genetics
|
2004
|
vol. 45
|
issue 2
225-236
EN
Due to the functions that estrogens play in the regulation of reproduction, development of the mammary gland, growth and differentiation of cells, estrogen receptors and their genes are considered candidates for the markers of production and functional traits in farm animals, including cattle. In the present study, on the basis of the sequences of the human, ovine, and porcine ER genes, available in the GenBank database, sets of PCR primers were designed and used to amplify the bovine ERa gene 5?-region. Seven overlapping fragments of the 5? region of the bovine ERa gene were amplified and then sequenced. Altogether, these fragments were composed in the 2853-bp sequence which was deposited in the GenBank database under accession no. AY340597. The sequenced fragment included the noncoding exons A, B, C, their putative promoters, and a part of the coding exon 1. A polymorphism within the 5? region of the bovine ERa gene ? A/G transition, which could be recognized with RFLP-BglI, lying upstream to the exon C, was identified for the first time using this sequence.
6

Transgenic animals in agriculture and medicine

64%
Rosochacki S. J., Zwierzchowski L.
Biotechnologia
|
1999
|
issue 2
7-24
EN
One of the most important issues in the use of transgenic technology in animal breeding is production of meat. For that purpose growth enhancing DNA sequences are introduced to the genome of pigs, sheep, cattle, rabbits and fish. The structural elements of the introduced genes usually GH, GRF or IGF-I, which are combined with the regulatory elements. In the case of transgenic swine, the higher growth rate, and the lower fat content were achieved by using these gene constructs. However, a number of such swines were sterile and had some other pathological problems. Similar and even more serious problems, were noticed in the case of transgenic sheep up. To date, most transgenic livestock projects focused on enhancing growth in farm animals by overexpressing growth hormone have to pathological changes in transgenic farm animals; only in transgenic fish no such problems have been ecountered. Fast growing transgenic trouts, carps and salmons carrying the so called 'all fish gene constructs' could be used even right now as food. Another goal in transgenic livestock projects is manufacturing of biologicaly active human proteins in the mammary gland. Transgenic sheep, goats, pigs and cows which produce human pharmaceuticals in their milk have been obtained. Some of such proteins undergo clinical trials. In another arena, it is planned to modify the milk of ruminants in order to obtain better product, which will be used in the dairy industry. It is possible to introduce more copies of milk protein genes into the genome, 'improving' the existing genes, or to inhibit the expression of some genes - thus reducing or eliminating the production of unwanted proteins. Another way to modify milk composition is the so called 'humanisation' of cow's milk by changing the proportion of cow's to human proteins. So far, all these modifications in the area of milk proteins are done mostly on laboratory animals and the introduction of these possibilities to farm animals would be desirable. Except for fish, transgenic farm animals are relatively safe to the environment; the probability of transgenic animals surviving and reproducing out of farms is rather very low. On the contrary, introduction of highly productive farm animals (cows, pigs) to breeding would be profitable to the environment and the called 'transgenic bioreactors' would also cause decrease of chemical or pharmaceutical industry contamination to the environment. For consumers, the products obtained from transgenic animals should be safe. 'Transgenic' products, which normaly exist in the nature, are not more unsafe than their natural counterparts. There is a clear consumer demand for testing all new products which normaly do not exist in the nature, i.e. proteins with totaly new amino acid sequence, before their introducing into the market. However, this is also valid for all novel food products, not only those obtained by biotechnology.
7

Structure, function, and polymorphism of the growth hormone receptor gene in humans and in animals

64%
Maj A., Zwierzchowski L.
Biotechnologia
|
2004
|
issue 1
85-107
EN
Growth hormone (GH) plays a central role in the regulation of growth and metabolism in animals and in humans. At the tissue levels, the pleiotropic actions of GH are mediated through their cell-surface receptor - GHR. The GHR belongs to the hematopoietic receptor superfamily. In mammals, GHR is the product of a single gene. In all studied, species GHR gene characterizes a complex structure of exon 1, coding for the 5'-untraslated region (5'-UTR). Several transcripts from the GHR gene were found differing by the presence of various length 5'-UTRs, resulting from the alternative splicing of the exon 1 fragments to a common splice site located 11-bp in the human and in bovine GHR gene exon 2. Numerous nucleotide sequence polymorphisms were found in the human GHR gene; some of them, those associated to GH resistance, were identified as the causative mutations of growth retardation, e.g. Laron's syndrome. In farm animals, genes coding for GH and GHR are obvious candidates for quantitative trait markers. Several polymorphic sites have been identified in the bovine GHR gene. At least in two cases, an association was reported between GHR gene polymorphism and performance traits. Detection of additional polymorphisms is necessary to help investigating the role of GHR variation in the production traits of the cattle. This article includes a review of literature on structure, function and polymorphism within GHR gene. Also, there are mentioned new data concerning the polymorphism recently identified by authors in the bovine GHR gene.
8

microRNA application to livestock

51%
Lisowski P., Goscik J., Zwierzchowski L.
Biotechnologia
|
2010
|
issue 3
115-125
EN
Post-transcriptional gene regulation guided by microRNAs has emerged as one of the major gene regulatory mechanisms in higher eukaryotes. microRNAs regulate gene translation through the recognition of complementary sequences between microRNAs and their target genes. Recent studies in livestock have revealed that many microRNAs are species- and tissue-specic, indicating that microRNAs play important roles in essential physiological processes in livestock, such as metabolism, and muscle and organ development. It is anticipated that many microRNAs will be linked to phenotypic differences or quantitative trait variations of livestock. The role of microRNA in developmental decisions that affect animal biology is of significant interest, yet the current literature on livestock models is limited. In this review, we summarize the current microRNA studies undertaken in livestock.
9

Biologically active recombinant human prolactin synthesised and secreted extracellularly in baculovirus expression system

51%
Michalik J., Zwierzchowski L., Strokovskaya L., Szolajska E.
Biotechnologia
|
2004
|
issue 1
260-273
EN
The recombinant human prolactin was synthesised as an extracellular protein expressed in baculovirus system. The concentration of prolactin in TC-100 medium was approximately 40 mg/l when the conditions of recombinant virus infection were properly chosen. The human prolactin present in culture medium was stable at 4C for several months up to one year. The recombinant product was a survival factor for the insect cells. In the presence of prolactin in the medium, the cells did not show any signs of lysis or disruption, which is in agreement with the view of the antiapoptotic action of prolactin. The results of Western-blot analysis showed similar ratio of glycosylated/non-glycosylated forms of the recombinant product to the hormone forms present in human physiological (osmiotic) fluids. The recombinant protein was biologically active as determined in mammary gland explant system. The recombinant hormone present in the culture media was shown to induce mRNAs for two milk proteins ? beta-casein and WAP in mammary explants cultured in the presence of insulin and hydrocortisone. The effect of the hormone was dose-dependant and the largest accumulation of both mRNAs was observed at rec-hPRL concentration of 0.1 mug/ml (approx. 4.3 x 10 -9 M). In this respect, the activity of the recombinant human prolactin was equal or even higher than that of bovine pituitary prolactin or human growth hormone.
10

Characterization of the CHORI-240 BAC clones containing the bovine CSN1S1, CSN2, STATH, CSN1S2 and CSN3 genes

51%
Sazanov A. A., Malewski T., Kaminski S., Zwierzchowski L.
Journal of Applied Genetics
|
2006
|
vol. 47
|
issue 3
243-245
EN
Nineteen BAC clones were identified by hybridization of the bovine genomic BAC library CHORI-240 with mixed CSN1S1- and CSN3-specific probes. Two of the clones were shown to contain the genes CSN1S1, CSN1S2, CSN2, STATH and CSN3, and five were proved to include the genes CSN2, STATH, CSN1S2 and CSN3. These data showed that the BAC contig was established for the whole casein cluster, including all known five genes.
11

Comparison of skeletal muscle transcriptional profiles in dairy and beef breeds bulls

45%
Sadkowski T., Jank M., Zwierzchowski L., Oprzadek J., Motyl T.
Journal of Applied Genetics
|
2009
|
vol. 50
|
issue 2
109-123
EN
A cDNA microarray (18 263 probes) was used for transcriptome analysis of bovine skeletal muscle (m. semitendinosus) in 12-month-old bulls of the beef breed Limousin (LIM) and the typical dairy breed Holstein-Friesian (HF, used as a reference). We aimed to identify the genes whose expression may reflect the muscle phenotype of beef bulls. A comparison of muscle transcriptional profiles revealed significant differences in expression of 393 genes between HF and LIM. We classified biological functions of 117 genes with over 2-fold differences in expression between the examined breeds. Among them, 72 genes were up-regulated and 45 genes were down-regulated in LIM vs. HF. The genes were involved in protein metabolism and modifications (22 genes), signal transduction (15), nucleoside, nucleotide and nucleic acid metabolism (13), cell cycle (9), cell structure and motility (9), developmental processes (9), intracellular protein traffic (7), cell proliferation and differentiation (6), cell adhesion (6), lipid, fatty acid and steroid metabolism (5), transport (5), and other processes. For the purpose of microarray data validation, we randomly selected 4 genes: trip12, mrps30, pycrl, and c-erbb3. Real-time RT-PCR results showed similar trends in gene expression changes as those observed in microarray studies. Basing on results of the present study, we proposed a model of the regulation of skeletal muscle growth and differentiation, with a principal role of the somatotropic pathway. It may explain at least in part the development of muscle phenotype in LIM bulls. We assume that the growth hormone directly or indirectly (through IGF-1) activates the calcium-signaling pathway with calcineurin, which stimulates myogenic regulatory factors (MRFs) and inhibits early growth response gene. The inhibition results in indirect activation of MRFs and impaired activation of TGF-beta1 and myostatin, which finally facilitates terminal muscle differentiation.
12

Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid

45%
Lisowski P., Pierzchala M., Goscik J., Pareek C. S., Zwierzchowski L.
Journal of Applied Genetics
|
2008
|
vol. 49
|
issue 4
367-372
EN
Expression patterns of candidate genes with important functions in animal metabolism can help to identify potential molecular markers for cattle production traits. Reverse transcription followed by polymerase chain reaction is a method for rapid and accurate mRNA quantification. However, for exact comparison of mRNA quantity in various samples or tissues, it is important to choose appropriate reference genes. In cattle, little information is available on the expression stability of housekeeping genes (HKGs). The aim of the present study is to develop a set of reference genes that can be used for normalization of concentrations of mRNAs of genes expressed in the bovine liver, kidney, pituitary and thyroid. The study was performed on 6-, 9-, and 12-month-old bulls of dairy and meat cattle breeds. Six HKGs were investigated: ACTB, GAPDH, HPRTI, SDHA, TBP, and YWHAZ. The most stably expressed potential reference HKGs differed among tissues/organs examined: ACTB, TBP, YWHAZ, GAPDH, HPRTI, and SDHA in the liver; GAPDH and YWHAZ in the kidney; GAPDH and SDHA in the pituitary; and TBP and HPRTI in the thyroid. The results showed that the use of a single gene for normalization may lead to relatively large errors, so it is important to use multiple control genes based on a survey of potential reference genes applied to representative samples from specific experimental conditions.
13

Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos

39%
Rosochacki S. J., Strzelecka M., Kozikova L., Matejczyk M., Oblap R., Poloszynowicz J., Zwierzchowski L.
Folia Biologica
|
2002
|
vol. 50
|
issue 1-2
61-67
EN
The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta -galactosidase (beta -gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were coinjected with the scaffold attachment sequences - SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta -galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta -galactosidase expression. The percentage of transgenesis with pCMV-lacZ alone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.
14

Polymorphism of the porcine growth hormone gene and its linkage to hormone gene and its linkage to microsatellites S0083 and S0090

39%
Korwin-Kossakowska A., Pierzchala M., Kuryl J., Zwierzchowski L., Cymerowska-Prokopczyk I., SiadkowskaE.
Journal of Applied Genetics
|
1999
|
vol. 40
|
issue 2
85-91
EN
Polymorphism of microsatellites S0083 and S0090 as well as of the second exon and second intron of the pGH (porcine growth hormone) gene was determined for 293 pedigrees obtained from crossing of 12 F1 (Zlotnicka Spotted x Polish Large White) boars with 64 F1 (Zlotnicka Spotted x Polish Large White) sows, experimental material arranged for a QTL mapping project. Microsatellites were genotyped by capillary electrophoresis of PCR products in an ABI PRISM 310 Genetic Analyser PERKIN-ELMER. The PCR-RFLP polymorphism of the GH gene was identified using HaeII and MspI restriction endonucleases. The following order of linked loci was established: GH-S0083-S0090. This is important information for the mapping of QTLs on chromosome 12.
15

Gene expression profiling in skeletal muscle of Holstein-Friesian bulls with single-nucleotide polymorphism in the myostatin gene 5'-flanking region

39%
Sadkowski T., Jank M., Zwierzchowski L., Siadkowska E., Oprzadek J., Motyl T.
Journal of Applied Genetics
|
2008
|
vol. 49
|
issue 3
237-250
EN
Myostatin (GDF-8) is a key protein responsible for skeletal muscle growth and development, thus mutations in the mstn gene can have major economic and breeding consequences. The aim of the present study was to investigate myostatin gene expression and transcriptional profile in skeletal muscle of Holstein-Friesian (Black-and-White) bulls carrying a polymorphism in the 5'-flanking region of the mstn gene (G/C transversion at position -7828). Real-time qRT-PCR and cDNA microarray revealed significantly lower mstn expression in muscle of bulls with the CC genotype, as compared to GG and GC genotypes. The direct comparison of skeletal muscle transcriptional profiles between the CC genotype and GG and GC genotypes resulted in identification of genes, of which at least some can be putative targets for myostatin. Using cDNA microarray, we identified 43 common genes (including mstn) with significantly different expression in skeletal muscle of bulls with the CC genotype, as compared to GG and GC genotypes, 15 of which were upregulated and 28 were downregulated in the CC genotype. Classification of molecular function of differentially expressed genes revealed the highest number of genes involved in the expression of cytoskeleton proteins (9), extracellular matrix proteins (4), nucleic acid-binding proteins (4), calcium-binding proteins (4), and transcription factors (4). The biological functions of the largest number of genes involved: protein metabolism and modification (10), signal transduction (10), cell structure (8), and developmental processes (8). The main identified signaling pathways were: Wnt (4), chemokines and cytokines (4), integrin (4), nicotine receptor for acetylocholine (3), TGF-beta (2), and cytoskeleton regulation by Rho GTPase (2). We identified previously unrecognized putatively myostatin-dependent genes, encoding transcription factors (EGR1, Nf1b, ILF1), components of the proteasomal complex (PSMB7, PSMD13) and proteins with some other molecular function in skeletal muscle (ITGB1BP3, Pla2g1b, ISYNA1, TNFAIP6, MST1, TNNT1, CALB3, CACYBP, and CTNNA1).
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