Primordial germ cells (PGCs) as a tool for creating transgenic chickens.

The transgenic chicken has great potential as a bioreactor for the production of valuable pharmaceutical proteins, notably in the oviduct/egg. Whereas conventional transgenic approaches have significant limitations in this species, an alternative approach employing primordial germ cells (PGCs), the progenitor cells to ova and spermatozoa, has now been successfully applied to the insertion of exogenous genes into birds. Recent developments in manipulating avian embryos make it possible to produce germline chimeras derived from transferred PGCs. In this review we describe the migration pathway of chicken PGCs during early development. We then summarize different methods for the isolation of PGCs and the diversity of techniques used to introduce genes into these cells. Finally, we describe an in vitro assay for testing tissue-specific vectors designed to express heterologous proteins in transgenic chickens.

Characterization of changes in the short unique segment of pseudorabies virus BUK-TK900 (Suivac A) vaccine strain.

Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900. We characterised changes in the U(S) region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300 bp, found in BamHI 7 fragment (covering most of the U(S) region) was located in the 28 K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass.

Application of FISH and Q-PCR techniques in breakpoint diagnostics in three cases of marker chromosomes derived from chromosome 15.

BACKGROUND: The goal of the study was a search for effective methods of diagnosing additional marker chromosomes. MATERIAL AND METHODS: Three cases of extra structurally abnormal chromosomes (ESACs) were diagnosed, the ESACs having been derived from chromosome 15 by cytogenetic techniques, the fluorescence in situ hybridisation (FISH) technique and the quantitative--polymerase chain reaction (Q-PCR). An application of a set of commercially available probes, specific for the 15q11.2-q12 regions (PWACR-Prader-Willi/Angelman Critical Region) allowed for a description of the breaking points. RESULTS: The presence of PWACR region was confirmed in one case and excluded in the other two. It was also attempted to apply the Q-PCR technique for a more accurate determination of the size of the region involved in chromosomal aberration, what would allow for a more reliable prognosing of the clinical outcome. In one of the patients, the breaking point was localized as distal to D15S144 locus, while it was proximal to D15S11 locus in the two remaining cases. CONCLUSIONS: The obtained results demonstrate a possibility of using the Q-PCR method in diagnosing unbalanced chromosome aberrations.

Successful DNA immunisation of rats against fasciolosis.

The liver fluke Fasciola hepatica contributes to great economic and health losses in the cattle industry in many countries, including Poland. Unfortunately, no vaccine against fasciolosis is commercially available. We have designed a DNA vaccine and tested it in rats. Groups of male or female rats received one intramuscular injection of 50 microg of a pcDNA 3.1 vector carrying cDNA encoding for a cysteine proteinase of F. hepatica. The plasmid was diluted in saline containing 0.05% bupivacaine. Control rats were injected with empty plasmid or not injected at all. All rats were challenged with 45 metacercariae of the fluke on day 28 of the experiment. Seven weeks after the challenge infection fluke burdens were evaluated in vaccinated and control rats. Male rats vaccinated with cysteine proteinase cDNA revealed 100% protection against F. hepatica infection. Females immunised in the same way exhibited the reduction of fluke burden by 74%.

Cloning of complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma.

Complementary DNA (cDNA) encoding a protein component pB1 (also pAIF-1 and DQH) of the 54-kilodalton glycoprotein of boar seminal plasma was cloned and its nucleotide sequence was determined (Gene Bank accession no. AF047026). The pB1 precursor protein is a 130-amino-acid-long polypeptide containing a 25-amino-acid-long signal peptide. The amino acid sequence of the pB1 is homologous to that of SFP1_BOVIN (named also BSP-A1/A2, PDC-109/ major protein and SVSp109), SFP3_BOVIN (BSP-A3), SFP4 BOVIN (BSP-30 KD), and SP1_HORSE (HSP-1) seminal plasma proteins. The homology extends also for the signal peptide of SFP1_BOVIN protein. All these seminal plasma proteins contain two fibronectin type-II domains that differ from those found in other proteins such as colagenases, fibronectins, and mannose receptors. The first domain located in the N-terminal region of pB1 is four amino acids shorter than those present in other proteins. High homology is also observed between 3' noncoding regions of the nucleotide sequences of cDNAs of pB1_PIG and SFP1_BOVIN (Gene Bank accession nos. AF047026 and P02784, respectively).

Fragments of LINE-1 retrotransposons flanked by inverted telomeric repeats are present in the bovine genome. Homology with human LINE-1 elements.

In the bovine genome we found two intrachromosomal DNA fragments flanked by inverted telomeric repeats (GenBank Accession Nos. AF136741 and AF136742). The internal parts of the fragments are homologous exclusively to the human sequences and to the consensus sequence of the L1MC4 subfamily of LINE-1 retrotransposons which are widespread among mammalian genomes. We found that distribution of homologous human sequences within our fragments is not random, reflecting a complicated pattern of insertion mechanisms of and maintenance of retrotransposons in mammalian genomes. One of the possible explanations of the origin of LINE-1 truncated elements flanked by inverted telomeric repeats in the bovine genome is that extrachromosomal DNA fragments may be modified by telomerase and subsequently, transferred into chromosomal DNA.

Effect of DNA-interacting drugs on phage T7 RNA polymerase.

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Effect of substance P and its precursor alpha-protachykinin on intracellular free calcium concentration in human polymorphonuclear leukocytes.

The increase in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation. The undecapeptide substance P can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, phagocytosis, and respiratory burst. In this study we investigated the ability of low-concentration (that can occur in vivo) substance P (10(-7) M) and its precursor alpha-protachykinin (3 x 10(-7) M) to increase the intracellular free calcium concentration in human polymorphonuclear leukocytes. Cells isolated from ten healthy donors were incubated with substance P or alpha-protachykinin in 1 mM calcium medium for 5 min and the intracellular free calcium concentration was monitored using the fluorescent calcium indicator Fura-2am. Polymorphonuclear leukocytes from 40% of donors responded to both agonists. The substance P- and alpha-protachykinin-induced increase in intracellular free calcium concentration was 59 +/- 13 nM and 58 +/- 12 nM and the extracellular calcium influx contributed to 87 +/- 8% and 54 +/- 8% of the calcium response, respectively. alpha-Protachykinin released almost all the calcium from intracellular stores, while substance P mobilized only 24 +/- 5% of this calcium pool. Finally, cells that responded to a single challenge with substance P and alpha-protachykinin were able to increase their intracellular free calcium concentration in response to each of three consecutive stimulations with these agonists. This may be an additional mechanism by which substance P and its precursor modify the function of human polymorphonuclear leukocytes.

Nucleotide sequence of c-H-ras-1 gene from B6C3F1 mice.

The c-H-ras-1 gene of an B6C3F1 mouse was isolated and nucleotide sequence determined. Our study has revealed that this c-H-ras-1 gene consists of four exons, separated by three introns ranging in size from 150 to 649 bp. The coding parts of the sequence of mouse c-H-ras-1 gene show no important differences as compared with those of the rat, hamster and human gene. More numerous changes were found in introns. The identity of mouse c-H-ras-1 gene with rat, hamster and human ones at the nucleotide level is 86.40%, 80.04% and 67.87%, respectively. Comparison of amino acids in protein sequence of c-H-ras gene of mouse, rat, hamster and human points to high degree of conservation of the gene.

[Etiology of chronic hepatitis as evaluated by liver biopsy and serum samples submitted to the Department of Immunopathology of the National Institute of Hygiene in 1993-1995]. / Etiologia przewleklych zapalen watroby na podstawie badan biopsji watroby i surowic nadeslanych do Zakladu Immunopatologii Panstwowego Zakladu Higieny w Latach 1993-1995.

The purpose of this study was to assess the relative incidence of chronic hepatitis in a population of patients with chronic liver disease and to determine the etiological spectrum of this syndrome with special reference to its defined histopathological forms. Histopathology aided by immunohistochemistry, and serology aided by the PCR method were employed in studies of liver biopsy specimens and serum samples, respectively. Out of 1150 patients with chronic liver disease examined, chronic hepatitis was diagnosed in 685 (60% of all cases examined). In this group, there were 308 males aged 18-74 yrs (mean 32 yrs), 153 females aged 18-71 yrs (mean 43 yrs), and 213 children aged 1-17 yrs (mean 8 yrs). Viral infections documented in these patients included HBV (50.4%), HCV (36.2%), HBV/HCV (7.2%) and HBV/HDV (0.7%); cryptogenic and autoimmune hepatitis (AIH) accounted for 2.9% and 2.6% all cases, respectively. In the group of minimal hepatitis (16.1%), HBV infection was documented in 66.4% of cases, HCV-in 29.1%, HBV/HCV-in 3.6% (one case of AIH was included into this group). In the group of mild hepatitis (44.2%), HBV infection accounted for 47.3% of cases, HCV-for 41.9%, HBV/HCV-for 9.9%, and 0.9% was diagnosed as cryptogenic. In the group of moderate hepatitis (19.6%), HBV infection accounted for 50% of cases, HCV-for 37.3%, and HBV/HCV-for 4.5%; cases of cryptogenic and AIH accounted for 3.7% and 4.5%, respectively. In the group of severe hepatitis (20.1%), HBV etiology was found in 44.9% of cases, HCV-in 28.3%, HBV/HCV-in 6.5% and HBV/HDV-in 3.6%; cryptogenic and AIH accounted for 6.5% and 8.0% of cases, respectively. There was a high incidence of low-titer autoantibodies (SMA, ANA and LKM) ranging from 75% in cryptogenic hepatitis and 51% in each HBV and HBV/HCV hepatitis to 46.3% in HCV hepatitis.

Bovine Alu-like sequences mediate transposition of a new site-specific retroelement.

We describe a new family of 3.1-kb repetitive sequences which is present in the bovine genome. The 5' and 3' ends of the unit are flanked with sequences homologous to the 5' and 3' halves of the bovine Alu-like monomer (BM), respectively. Distribution of the 5' ends of the family members in the genome is not random. They are close to the truncated bovine Alu-like dimer (BD) which, in some cases, is followed by 40-bp repeated sequences containing block A of the RNA polymerase III promoter. The ORFs found within the unit code for peptides homologous to amino-acid sequences characteristic for reverse transcriptases (RT). The family members may be considered as mutant mobile elements whose propagation in the genomes was accomplished by means of a process including site-specific recognition with BD. Because of this, we call this family the bovine dimer-driven family (BDDF).

Expression and characterization of the multiplied, recombinant preS1 antigen of hepatitis B virus.

The amino acid sequence encoded by the preS1 region of hepatitis B virus genome is expressed on the surface of virions and subviral particles. The preS1 region is involved in the recognition of specific receptors responsible for the attachment of HBV to the host cell. The cell receptor binding site was assigned to the preS1 (20-47 aa) fragment. In order to obtain a large quantity of preS1 binding domains of HBV the expression vector pWX4 was constructed. It contains four tandemly joined DNA sequences, each coding for preS1 (20-49 aa), fused with the 3' end of a DNA fragment coding for 450 aa of beta-galactosidase. E. coli cells transformed with this vector produce fusion protein beta-gal-preSlx4 in the form of inclusion bodies. Owing to the specific trypsin digestion, the preSlx4 domain was cleaved from the fusion protein. The resulting product, a 16 kDa protein, was isolated and purified by anion exchange chromatography. The presence of four Asp-Pro bonds in this sequence and the primary structure of the first 28 N-terminal amino acids were determined. Following the confirmation of the antigenic properties, the recombinant preS1 protein was used for detection of the anti-preS1 response in sera from HBV infected patients.

Antibodies to recombinant fragment 212-276 of protein C specifically recognize the intact human molecule.

The peptide fragment Pro212-Ile276 of human protein C was produced as a part of a fusion protein in Escherichia coli. The identity of the peptide was confirmed by immunoblotting experiments using specific antibodies to intact protein C. The peptide Pro212-Ile276 was isolated from the fusion protein after mild hydrolysis with formic acid by gel filtration and reverse-phase HPLC. This peptide fragment was used to produce antibodies specific for the heavy chain of protein C which recognized native protein C present in blood plasma. Antibodies to intact protein C reacted also with the Pro212-Ile276 peptide fragment, indicating that this region is immunogenic in intact protein C and may represent a native epitope.

Synthesis of a gene encoding bovine substance P precursors and its expression in Escherichia coli.

Using an efficient Escherichia coli expression system, we have been able to obtain the precursor of substance P, alpha-preprotachykinin (alpha PPT). The alpha PPT protein is produced in E. coli as a fusion to beta-galactosidase, and accumulates in the cytoplasm as insoluble inclusion bodies. We also produced protachykinin (alpha PT), i.e., alpha PPT without a signal peptide. Further purification and characterization of the alpha PPT and alpha PT polypeptides strongly suggest that fully purified products can be obtained using our procedures.

Expression of multivalent pre-S1 antigen of hepatitis B virus in Escherichia coli (synthetic oligodeoxynucleotides, surface antigens, recombinant DNA, fusion protein, beta-galactosidase).

The nucleotide sequence encoding 30 amino acids (aa) of the pre-S1 envelope region of the human hepatitis B virus has been constructed from twenty chemically synthesized oligodeoxynucleotides by simultaneous ligation. The DNA fragment containing four repeated sequences encoding the pre-S1 region (aa 20-49) has been inserted into the lacZ gene of the plasmid pWR450.1, yielding the recombinant pWX4 plasmid. The Escherichia coli DH5 strain transformed with pWX4 produces a beta-galactosidase-[-pre-S1(20-49) x 4] fusion protein. The hybrid protein containing 127 aa of repeated pre-S1 region has been isolated from Escherichia coli as inclusion bodies and purified by anion exchange chromatography. The antigenic properties of this fusion protein were confirmed by immunoblotting with pre-S1-specific monoclonal antibodies.

A new family of dispersed, highly repetitive sequences in bovine genome.

A new family of highly repetitive sequences which are dispersed in bovine genome is described. The members of the family are visible on agarose or polyacrylamide gels as a diffused band about 510 bp in length arising after digestion with PstI restriction nuclease. This family of fragments comprises the 160 bp bovine Bsu family and is linked with bovine Alu-like sequences.

Nucleotide sequence of bovine 1.723 satellite DNA.

The nucleotide sequence of the bovine 1.723 satellite DNA repeated unit was determined. The 680 bp long period of this satellite DNA does not show any significant sequence similarities with the known bovine satellite DNAs. Short repetitive sequences which are parts of 680 bp long repeated units do not form any orderly periodical structure. It seems, however, that the basic repeated unit of the 1.723 bovine satellite DNA has been formed by successive duplications of two, about 100 bp long sequences. The sequence divergence between different copies of the 680 bp repeated unit was also analyzed.