The Oncogenic MicroRNA OncomiR-21 Overexpressed during Marek's Disease Lymphomagenesis Is Transactivated by the Viral Oncoprotein Meq

Gallid herpesvirus 2 (GaHV-2) is an oncogenic herpesvirus that causes T lymphoma in chicken. GaHV-2 encodes a basic leucine zipper (bZIP) protein of the AP-1 family, Meq. Upon formation of homo- or heterodimers with c-Jun, Meq may modulate the expression of viral and cellular genes involved in lymphomagenesis. GaHV-2 also encodes viral microRNAs (miRNAs) involved in latency and apoptosis escape. However, little is known about cellular miRNA deregulation during the development of GaHV-2-associated lymphoma. We determined the cellular miRNA expression profiles of chickens infected with a very virulent strain (RB-1B) or a vaccine strain (CVI988) or noninfected. Among the most deregulated cellular miRNAs, we focused our efforts on gga-miR-21, which is upregulated during GaHV-2 infection. We mapped the gga-miR-21 promoter to the 10th intron of the TMEM49 gene and found it to be driven by AP-1- and Ets-responsive elements. We show here that the viral oncoprotein Meq binds to this promoter, thereby transactivating gga-miR-21 expression. We confirmed that this miRNA targets chicken programmed death cell 4 (PDCD4) and promotes tumor cell growth and apoptosis escape. Finally, gga-miR-21 was overexpressed only during infection with a very virulent strain (RB-1B) and not during infection with a nononcogenic strain (CVI988), providing further evidence for its role in GaHV-2 lymphomagenesis. Our data therefore suggest an additional role for Meq in GaHV-2-mediated lymphomagenesis through the induction of miR-21 expression.     

Microsatellite Instability in Marek's Disease Virus Infected Primary Chicken Embryo Fibroblasts

ABSTRACT: BACKGROUND: Marek's disease virus (MDV), an oncogenic alpha-herpesvirus, causes a devastating disease in chickens characterized by development of lymphoblastoid tumors in multiple organs. Microsatellite instability (MSI), a symptom of defect in DNA mismatch repair function, is a form of genomic instability frequently detected in many types of tumors. However, the involvement of MSI in MDV-infected cells has not been investigated. In this study, we determined the presence and frequency of MSI in primary chicken embryo fibroblasts infected with or without virulent RB-1B strain of MDV in vitro. RESULTS: 118 distinct microsatellite markers were analyzed by polymerase chain reaction (PCR) in 21 samples. MSI was found in 91 of 118 markers, and 12 out of 118 demonstrated frequency of MSI at [greater than or equal to] 40%. 27 of 118 microsatellite loci did not show microsatellite instability. CONCLUSIONS: These findings showed that MSI was a real event occurring in primary chicken embryo fibroblasts infected with MDV in vitro as evidenced by the high frequency of MSI, and may be specifically associated with genome alteration of host cells during MDV infected.     

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.     

Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.     

Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography

Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI⁺). MSI is detected in about 15 % of all CRCs; 3 % are of these are associated with Lynch syndrome and the other 12 % are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI⁺ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100 % in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.     

Molecular characterization of the duck enteritis virus <Emphasis Type="Italic">UL4</Emphasis> gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.      

应用荧光标记多重PCR对原发性胃癌进行微卫星不稳定性检测

为探讨 19号染色体微卫星不稳定性 (microsatelliteinstability ,MSI)在原发性胃癌发生中的作用 ,选择覆盖 19号染色体的 2 6个高分辨 (<5cM)微卫星标记 ,采用FAM、TET和HEX3种不同颜色的荧光染料标记微卫星引物 ,应用荧光标记多重PCR对 44例原发性胃癌及其正常对照组织进行扩增 ,产物在ABI3 77测序仪上经变性聚丙烯酰胺凝胶电泳 ,通过GeneScanTM及GenotyperTM 软件进行图像收集和MSI分析。同时采用PCR SSLP 银染技术检测了其中4个位点的MSI状态。 2 6个微卫星位点中 ,2 2个 (84 62 % )存在MSI,以D19S40 2MSI发生频率最高 (2 2 73 % ) ,平均MSI频率为 8 3 %。 44例胃癌中 ,3 4例 (77 2 7% )在一个或一个以上位点出现MSI。MSI作为胃粘膜癌变过程中一种可能的分子病理学机制 ,可作为胃癌分子检测的指标之一     

Latent Marek's disease virus can be activated from its chromosomally integrated state in herpesvirus-transformed lymphoma cells.

Marek's disease virus (MDV), a lymphotropic herpesvirus, induces T-cell lymphomas in chicken, its natural host. The lymphoma cells are latently infected with MDV but the viral contribution to the transformed phenotype is not understood. To investigate the virus-cell interaction, we focused on the status of MDV in the transformed cells. By the use of highly sensitive fluorescent in situ hybridization with metaphase chromosomes, we found (i) MDV DNA to be randomly integrated at multiple sites in the chromosomes of primary lymphoma cells from chicken tissues; (ii) extrachromosomal, circular MDV genomes were absent and linear virion DNA was usually not detectable in the latently infected lymphoma cells; (iii) the pattern of integration sites revealed the clonal origin of the tumour cells; which (iv) was retained in in vitro established cell lines derived from primary lymphomas; (v) activation of the lytic phase of MDV's life cycle occurred in vitro suggesting that MDV can escape from its integrated status by an unknown mechanism.     

结直肠腺瘤中的微卫星不稳定性类型

应用微切割 聚合酶链反应 单链长度多态性 (PCR SSLP)的方法 ,检测 1 6个微卫星位点在 5 9例 6 2个结直肠腺瘤标本的微卫星不稳定性状态 .结果表明 :腺瘤 1 6个位点的总微卫星不稳定性(microsatelliteinstability ,MSI)发生率为 1 4 4 % ,MSI H所占的比率为 9 7% ;在 1 0例可以同时微切割得到腺瘤和癌变成分的病例中 ,腺瘤和癌变成分在每个微卫星位点的改变情况不完全相同 ,并且当在某一位点同时表现为阳性时 ,部分凝胶电泳的图像相同 ,而部分不同 ;在某些位点表现为癌变成分的异常条带泳动速度更快 ,说明序列比腺瘤中更短 ;MSI H与病人的年龄、性别、腺瘤发生部位和病理学亚型之间未见统计学差异 ,但MSI H组的平均年龄 (5 6 5 0± 1 1 38)低于MSI L组 (6 0 36±1 1 34) ,女性所占比率 (5 6 )明显高于男性 ,6例MSI H中无 1例组织学类型为管状腺瘤 ;各位点在MSI H组的MSI改变率明显高于MSI L组 ,在TGFβRⅡ (A) 1 0 、hMSH6、TCF4、BAT2 6等位点有明显差异 (P <0 0 5 ,其中BAT2 6的P <0 0 1 ) .可以推断 :在结直肠癌发生发展的早期即腺瘤阶段即可表现微卫星不稳定性 ;微卫星不稳定性可以随结直肠肿瘤的发展过程而发展 ,并且特定的微卫星位点的改变可能仅发生于肿瘤进程的特定阶段 ;在结直肠癌     

Identification of the neoplastically transformed cells in Marek's disease herpesvirus-induced lymphomas: recognition by the monoclonal antibody AV37

Understanding the interactions between herpesviruses and their host cells and also the interactions between neoplastically transformed cells and the host immune system is fundamental to understanding the mechanisms of herpesvirus oncology. However, this has been difficult as no animal models of herpesvirus-induced oncogenesis in the natural host exist in which neoplastically transformed cells are also definitively identified and may be studied in vivo. Marek's disease (MD) herpesvirus (MDV) of poultry, although a recognized natural oncogenic virus causing T-cell lymphomas, is no exception. In this work, we identify for the first time the neoplastically transformed cells in MD as the CD4(+) major histocompatibility complex (MHC) class I(hi), MHC class II(hi), interleukin-2 receptor alpha-chain-positive, CD28(lo/-), phosphoprotein 38-negative (pp38(-)), glycoprotein B-negative (gB(-)), alphabeta T-cell-receptor-positive (TCR(+)) cells which uniquely overexpress a novel host-encoded extracellular antigen that is also expressed by MDV-transformed cell lines and recognized by the monoclonal antibody (MAb) AV37. Normal uninfected leukocytes and MD lymphoma cells were isolated directly ex vivo and examined by flow cytometry with MAb recognizing AV37, known leukocyte antigens, and MDV antigens pp38 and gB. CD28 mRNA was examined by PCR. Cell cycle distribution and in vitro survival were compared for each lymphoma cell population. We demonstrate for the first time that the antigen recognized by AV37 is expressed at very low levels by small minorities of uninfected leukocytes, whereas particular MD lymphoma cells uniquely express extremely high levels of the AV37 antigen; the AV37(hi) MD lymphoma cells fulfill the accepted criteria for neoplastic transformation in vivo (protection from cell death despite hyperproliferation, presence in all MD lymphomas, and not supportive of MDV production); the lymphoma environment is essential for AV37(+) MD lymphoma cell survival; pp38 is an antigen expressed during MDV-productive infection and is not expressed by neoplastically transformed cells in vivo; AV37(+) MD lymphoma cells have the putative immune evasion mechanism of CD28 down-regulation; AV37(hi) peripheral blood leukocytes appear early after MDV infection in both MD-resistant and -susceptible chickens; and analysis of TCR variable beta chain gene family expression suggests that MD lymphomas have polyclonal origins. Identification of the neoplastically transformed cells in MD facilitates a detailed understanding of MD pathogenesis and also improves the utility of MD as a general model for herpesvirus oncology.     

Japanese quail microsatellite loci amplified with chicken-specific primers

Forty-eight primer pairs for chicken (Gallus gallus) microsatellite loci were tested in polymerase chain reaction (PCR) amplification of Japanese quail (Coturnix japonica) genomic DNA. Amplification products were obtained from 28 primer-pairs (58.3%) after optimizing the PCR conditions. Eleven (22.9%) of these generated specific products and 17 yielded non-specific amplification products. Eight markers (ADL0037, ADL0038, ADL0142, ADL0143, ADL0206, ADL0315, ADL0366, and HUJ0006) were polymorphic and three were monomorphic (ADL0023, ADL0024, and ADL0257) in four Japanese quail populations. Specific amplification products from each of the 11 PCR primers were sequenced. Seven of the eight polymorphic and one of three monomorphic markers contained simple tandem repeats. Six of these microsatellite loci (ADL0037, ADL0315, ADL0142, ADL0143, ADL0366 and ADL0257) may be homologous to the corresponding chicken loci from which the markers were developed.     

A genetic and cytogenetic map for the duck (Anas platyrhynchos)

A genetic linkage map for the duck (Anas platyrhynchos) was developed within a cross between two extreme Peking duck lines by linkage analysis of 155 polymorphic microsatellite markers, including 84 novel markers reported in this study. A total of 115 microsatellite markers were placed into 19 linkage groups. The sex-averaged map spans 1353.3 cM, with an average interval distance of 15.04 cM. The male map covers 1415 cM, whereas the female map covers only 1387.6 cM. All of the flanking sequences of the 155 polymorphic loci--44 monomorphic loci and a further 41 reported microsatellite loci for duck--were blasted against the chicken genomic sequence, and corresponding orthologs were found for 49. To integrate the genetic and cytogenetic map of the duck genome, 28 BAC clones were screened from a chicken BAC library using the specific PCR primers and localized to duck chromosomes by FISH, respectively. Of 28 BAC clones, 24 were detected definitely on duck chromosomes. Thus, 11 of 19 linkage groups were localized to 10 duck chromosomes. This genetic and cytogenetic map will be helpful for the mapping QTL in duck for breeding applications and for conducting genomic comparisons between chicken and duck.     

食管鳞癌MLH1基因改变及其与微卫星不稳定的关系

MLH1是位于3p21．3上的一个DNA错配修复基因,其异常与多种肿瘤相关。为探索食管鳞癌中MLH1基因改变及其与微卫星不稳定(MSI)的关联情况,采用微卫星分析和RT—PCR方法检测了14个微卫星标志在食管癌中的状况及MLH1转录水平的表达,发现35％的食管癌出现至少一个微卫星的不稳定,66．7％的肿瘤在MLH1基因内标志D3S1611位点表现为杂合性丢失,但是MLH1没有明显的mRNA表达下调。MSI与食管癌分期、分级、淋巴结转移、患者年龄和性别等参数及MLH1基因杂合性丢失(LOH)之间无统计学意义的相关性。这些结果表明：食管癌中MLH1存在较高频率的等位基因丢失,但其mRNA表达水平并无明显异常;所测微卫星标志的不稳定是食管癌的频发事件,与MLH1基因LOH不存在必然联系。     

Detection of a Novel Bovine Lymphotropic Herpesvirus

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.     

Differentiating large cell lymphoma from indolent small B-cell lymphoma in fine needle aspirates using p53, PCNA and transformed lymphocyte count

OBJECTIVE: To determine the usefulness of proliferating cell nuclear antigen (PCNA), p53 protein expression and transformed lymphocyte count (TLC) as adjunctive tests to differentiate indolent small B-cell lymphoma from large cell lymphoma in fine needle aspiration biopsies. STUDY DESIGN: Aspirates of lymphoproliferative disorders from April 1993 to January 1997 were reviewed. The percentage of TLCs was determined on the Papanicolaou smear. The percentage and intensity of p53 and PCNA immunocytochemical staining was evaluated on cell block sections. These results were compared and correlated with the final diagnoses based on available morphology, flow cytometry and clinical history. RESULTS: There were 40 cases of non-Hodgkin's lymphoma and 12 reactive lymph nodes. Adequate cell blocks were available on 16 large cell lymphomas, 7 grade 1-2 follicular center cell lymphomas, 6 mucosal associated lymphoid tissue lymphomas, 2 small lymphocytic lymphomas and 2 mantle cell lymphomas. Average TLC and p53 nuclear staining was highest in large cell lymphomas (57% TLC and 24% p53), followed by grades 1 and 2 follicular lymphomas (14% TLC and 15% p53) and lowest in other indolent lymphomas (< 10% TLC and < 1% p53). Average PCNA staining was highest in large cell lymphomas (46%) and lowest in small lymphocytic lymphomas (7%); however, TLC was the best parameter for differentiating large cell lymphoma from indolent small B-cell lymphoma. CONCLUSION: TLC differentiated large cell lymphoma from indolent small B-cell lymphoma better than either p53 or PCNA alone or in combination. Significant overlap between categories limits usefulness of these immunocytochemical stains for differentiating these entities.     

Microsatellite Alterations Are also Present in the Less Aggressive Types of Adult T-Cell Leukemia-Lymphoma

Background

Adult T-cell leukemia/lymphoma (ATL) is a mature T-cell neoplasia etiologically linked to HTLV-1. Manifestations of ATL are diverse and different clinical types with different tissue involvement and aggressiveness have been described. The mechanisms that lead to the development of ATL clinical types have not yet been clarified. Considering that in ATL patients HTLV-1 infection generally occurs in childhood, a multistep carcinogenesis model has been proposed. Microsatellite alterations are important genetic events in cancer development and these alterations have been reported in the aggressive types of ATL. Little is known about oncogenesis of the less aggressive types.

Methodology/Principal Findings

In this study we investigated the role of the microsatellite alterations in the pathogenesis mediated by HTLV-1 in the different types of ATL. We examined the presence of microsatellite instability (MSI) and loss of heterozigosity (LOH) in matched pair samples (tumoral and normal) of 24 patients with less aggressive types (smoldering and chronic) and in aggressive types (acute and lymphoma) of ATL. Four microsatellite markers D10S190, D10S191, D1391 and DCC were analyzed. MSI was found in four patients, three smoldering and one chronic, and LOH in four patients, three smoldering and one acute. None of the smoldering patients with microsatellite alterations progressed to aggressive ATL.

Conclusions/Significance

To our knowledge, this is the first report describing the presence of MSI and LOH in the less aggressive types of ATL. These results indicate that microsatellite alterations may participate in the development of the less aggressive types of ATL.     

Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas

Although information on the molecular pathways in radiation carcinogenesis is accumulating, the data are still relatively scanty. To find the tumor suppressor locus associated with radiation carcinogenesis, we determined the frequency and distribution of loss of heterozygosity (LOH) of X-ray-induced thymic lymphomas of B6C3F(1) mice using 58 microsatellite markers and compared the results with those for spontaneous lymphomas and N-ethylnitrosourea (ENU)-induced lymphomas. Based on the results, we describe a unique locus with frequent LOH in the centromeric region of chromosome 11 of X-ray-induced lymphomas. This locus has never been observed to be altered similarly in either ENU-induced or spontaneous lymphomas, suggesting radiation-specific molecular alteration. The LOH patterns of individual thymic lymphomas indicated that the common region of LOH was located within 1.6 cM between D11Mit62 and D11Mit204, a region syntenic to human chromosome 7p13. Linkage analysis revealed that the markers of the common LOH region were genetically linked to Ikaros (now known as Znfn1a1), a master gene of lymphopoiesis. Although the presence of radiation-associated LOH in other loci cannot be ruled out, these results suggest a novel molecular pathway in induction of thymic lymphomas by ionizing radiation.     

A genome-wide set of 106 microsatellite markers for the blue tit (Cyanistes caeruleus)

We have characterized a set of 106 microsatellite markers in 26-127 individual blue tits (Cyanistes caeruleus), and assigned their location on the zebra finch (Taeniopygia guttata) and on the chicken (Gallus gallus) genome on the basis of sequence homology. Thirty-one markers are newly designed from zebra finch EST (expressed sequence tags) sequences, 22 markers were developed by others from EST sequences using different methods and the remaining 53 loci were previously designed or modified passerine markers. The 106 microsatellite markers are distributed over 26 and 24 chromosomes in the zebra finch and in the chicken genome respectively and the number of alleles varies between 2 and 49. Eight loci deviate significantly from Hardy-Weinberg equilibrium and show a high frequency of null alleles, and three pairs of markers located in the same chromosome appear to be in linkage disequilibrium. With the exception of these few loci, the polymorphic microsatellite markers presented here provide a useful genome-wide resource for population and evolutionary genetic studies of the blue tit, in addition to their potential utility in other passerine birds.     

Diagnosis of B-cell lymphoma. Utility of the polymerase chain reaction for detecting clonality from archival cytologic smears

OBJECTIVE: To apply the polymerase chain reaction (PCR) to detect clonality for potentially helping to establish a definitive diagnosis of lymphoma in cytologic material. STUDY DESIGN: In this retrospective study, Papanicolaou-stained cytologic smears and formalin-fixed, paraffin-embedded tissues from 17 cases of B-cell lymphoma were examined to investigate their clonality by a PCR technique using three different approaches (FR3, FR3A and FR2) for amplification of immunoglobulin heavy chain genes. Cytologic smears from 10 cases of nonneoplastic lymphoid tissues and T-cell lymphomas served as negative controls. RESULTS: Monoclonality was detected in 9 of 17 cases (53%) of B-cell lymphoma in cytologic smears as compared with 8 of 16 cases (50%) in tissue sections. Semi-nested PCRs (FR3A/FR2) were superior to the single PCR (FR3) in the detection rate (41% vs. 18%). Five of seven cases (71%) of marginal zone B-cell lymphomas showed monoclonality, whereas only 4 of 10 cases (40%) of diffuse large B-cell lymphomas did so. Monoclonality was demonstrated in none of the negative controls. CONCLUSIONS: Clonality detection in B-cell lymphomas by PCR using cytologic smears is specific and equal in sensitivity to that using formalin-fixed, paraffin-embedded tissues. The detection rate is especially excellent in marginal zone B-cell lymphoma, in which the cytologic diagnosis is particularly challenging. Combined seminested PCRs for FR3A and FR2 are advocated for a reliable assessment of clonality.     

Use of chicken microsatellite markers in turkey: a pessimistic view

Eighty-eight chicken microsatellite markers, previously developed in our laboratory, were tested for their ability to amplify polymorphic fragments using turkey genomic DNA. Amplification products were obtained for 61 chicken microsatellite markers (69.1%) whereas 27 (30.9%) did not give rise to any products, even when different polymerase chain reaction conditions were employed. From the 61 markers that gave a product, only eight showed a length polymorphism while 37 were monomorphic on the three divergent commercial turkey lines used. The remaining 16 markers yielded many unspecific bands and no specific amplification product could be obtained. Five polymorphic and eleven monomorphic products contained a detectable microsatellite repeat. Furthermore, of the markers that detected a polymorphism in turkey, the observed heterozygosity (15–50%) and allelic variation (only 2 in most cases) was very low. Therefore, on the basis of our results, we think that chicken microsatellite markers are not very useful for mapping purposes in turkey.