A PCR-based test for the presence of endogenous virus gene evA in chickens

An endogenous virus, denoted ev A, is present at high frequency in all brown egg layer lines. Using inverse polymerase chain reaction (PCR) based on the viral LTR regions, products were obtained containing cellular sequences 5' and 3' to the viral insertion point. PCR of chicken genomic DNA was carried out, using primers chosen from the 5' and 3' cellular sequences and a primer chosen from either the U3 or U5 portions of the viral LTR. Amplification of DNA from birds that did not carry ev A with the primer triplets always gave a single 364bp reaction product, interpreted as representing the flank-to-flank amplification product. Amplification of DNA from known homozygous or heterozygous ev A carriers, with the same primer triplets, always gave both the expected junction product and 364bp product. Therefore, these primer sequences can be used to distinguish ev A carriers from non-carriers but cannot distinguish between homozygous and heterozygous ev A carriers.     

Haplotypic variation and characteristics across the toll-like receptor 3 locus in chickens

Toll-like receptor 3 (TLR3) has an important protective function against viral infection. The ability of an individual to respond properly to TLR ligands may be impaired by variants located in the TLR genes. By directly PCR sequencing four exons and their flanking sequence of chicken TLR3, a total of 50 nucleotide variants were identified from five breeds. Tibetan chickens and Silkies exhibited more abundant variation sites and rare alleles. Thirty haplotypes were reconstructed, with 31 variants whose minor allelic frequency was above 5% in five breeds, which revealed four divergent clades. Chicken TLR3 was partitioned into three haplotype blocks by the htSNPer program, and six tag SNPs could be used to distinguish these 30 haplotypes. Thirty variants were located in the coding sequence of chicken TLR3, and 16 of them were non-synonymous substitutions. It is predicted that p.Ser180Gly amino substitution could form an N-myristoylation site; the p.Lys240Thr amino substitution in chicken TLR3 could result in the loss of one protein kinase C phosphorylation site. These data provide a basic understanding of chicken TLR3 sequence variation and provide haplotypic markers for disease association studies.     

Tail-to-Head Arrangement of a Partial Chicken Glyceraldehyde-3-Phosphate Dehydrogenase Processed Pseudogene

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5′ region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization. Received: 15 November 1996 / Accepted: 1 April 1997     

Characterization of CR1 repeat random PCR markers for mapping the chicken genome

Polymerase chain reaction (PCR) primers complementary to portions of the chicken repetitive element CR1 have been used previously to generate useful markers on the chicken genome linkage map. To understand better the genetic basis for this technique and to convert CR1–PCR loci to markers useful in physical genome mapping, five polymorphic CR1–PCR-generated DNAs were cloned and partially sequenced. Inverse PCR was then employed to clone the corresponding region of the genomes of both the Jungle Fowl (JF) and White Leghorn (WL) parental DNA templates. Our results demonstrate that some of the CR1–PCR-generated DNAs arise from priming at an endogenous CR1 element, whereas others are due to chance complementarity between the CR1–PCR primer in use and random annealing sites in the genome, unrelated to a demonstrable CR1 element. In all five instances, it was possible to identify the sequence difference between the JF and WL parental DNAs that gave rise to the initial polymorphism and design allele-specific PCR primer sets that uniquely detect that polymorphism. In four of the five instances, the polymorphism was a one or two basepair sequence difference within the primer annealing site, but in the fifth case the responsible difference was outside, but very close to, the annealing site. In all instances the allele-specific PCR for the sequence polymorphism mapped identically with the corresponding CR1–PCR amplification polymorphism. We conclude that CR1–PCR provides an efficient and reliable mechanism for genome mapping in avians that can correlate linkage and physical mapping approaches.     

Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC<,10200> strain).     

利用长引物嵌套式反向PCR方法克隆雅致枝霉Δ6-脂肪酸脱氢酶基因上游序列

用限制酶EcoRⅠ、KpnⅠ分别对雅致枝霉As3.2806基因组DNA进行消化,而后在低浓度条件下利用T4DNA连接酶使DNA自身环化。根据已知基因序列,设计一对长度为35nt的长反向引物和两对较短的引物,以基因组连接产物为模板,通过三轮嵌套式PCR反应,获得一长度约为4kb的扩增片段。经序列测定表明得到了Δ6-脂肪酸脱氢酶基因上游序列约为1.3kb,初步序列分析显示该序列为一潜在的启动子序列。     

Technical advance: single pollen typing combined with laser-mediated manipulation

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.     

CArG, CCAAT, and CCAAT-like protein binding sites in avian retrovirus long terminal repeat enhancers.

A strong enhancer element is located within the long terminal repeats (LTRs) of exogenous, oncogenic avian retroviruses, such as Rous sarcoma virus (RSV) and the avian leukosis viruses. The LTRs of a second class of avian retroviruses, the endogenous viruses (evs), lack detectable enhancer function, a property that correlates with major sequence differences between the LTRs of these two virus groups. Despite this lack of independent enhancer activity, we previously identified sequences in ev LTRs that were able to functionally replace essential enhancer domains from the RSV enhancer with which they share limited sequence similarity. To identify candidate enhancer domains in ev LTRs that are functionally equivalent to those in RSV LTRs, we analyzed and compared ev and RSV LTR-specific DNA-protein interactions. Using this approach, we identified two candidate enhancer domains and one deficiency in ev LTRs. One of the proposed ev enhancer domains was identified as a CArG box, a motif also found upstream of several muscle-specific genes, and as the core sequence of the c-fos serum response element. The RSV LTR contains two CArG motifs, one at a previously identified site and one identified in this report at the same relative location as the ev CArG motif. A second factor binding site that interacts with a heat-stable protein was also identified in ev LTRs and, contrary to previous suggestions, appears to be different from previously described exogenous virus enhancer binding proteins. Finally, a deficiency in factor binding was found within the one inverted CCAAT box in ev LTRs, affirming the importance of sequences that flank CCAAT motifs in factor binding and providing a candidate defect in the ev enhancer.     

Expression of endogenous retrovirus ev/J <Emphasis Type="Italic">gp85</Emphasis> gene and analysis of its immunoreactivity in comparison with exogenous viral protein

The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC₁₀₂₀₀ strain). Foundation items: Natural Science Foundation of China (30460098); China Postdoctoral Science Foundation funded project (2005038585).     

Isolation of recombinant field strains of Marek's disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

鸡端粒酶RNA基因的克隆

本研究采用扩增条件优化的PCR扩增技术,以MDCC-MSBl细胞基因组DNA为模板扩增出鸡端粒酶RNA(chicken telomerase RNA,chTR)全长基因,克隆到pMD18-T载体中,经酶切鉴定和PCR鉴定后测定序列.序列分析表明所克隆的鸡端粒酶RNA基因全长465 bp,其中模板区的11个核苷(5'-CUAACCCUAAU-3')合成端粒亚单位(TTAGGG)n.chTR基因的克隆为进一步研究chTR在马立克氏病发病过程中的作用以及马立克氏病的发病机制提供可能的序列基础.     

Genome structure and expression of the ev/J family of avian endogenous viruses.

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157-10164, 1998). Based on the fact that the endogenous subgroup J-related env genes were associated with long terminal repeats (LTRs), we concluded that these LTR-env sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the pol gene and various amounts of gag and env gene sequences. Sequencing of the gag genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J gag gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Cloning of the Promoter Regions of Mouse TGF-{beta} Receptor Genes by Inverse PCR with Highly Overlapped Primers

In order to isolate promoters of mouse TGF-ß receptorgenes, we used inverse PCR with highly overlapped primers correspondingto the 5' sequence of the receptor cDNAs. Nested primer setsonly covered a 30- to 40-base region of the sequences. HinfI-digestedand self-ligated mouse genomic DNA was used as a PCR template.Only one band for each receptor was seen after PCR. The amplifiedDNA fragments could direct luciferase production when the luciferasecoding sequence was ligated after the fragments. The sequenceof the fragment which correspond to the type II receptor showedpartial homology with the promoter region of the human TGF-ßtype II receptor. Thus, the inverse PCR with highly overlappedprimers could be an easy way to isolate the promoter regionsof many genes.