Genome-wide scan reveals divergent selection among taurine and zebu cattle populations from different regions.

In this study, to identify genomic signatures of divergent selection, we genotyped 10 cattle breeds/populations (n = 275), representing eight Ethiopian cattle populations (n = 229) and two zebu populations (n = 46) adapted to tropical and sub-tropical environments, using the high-density single-nucleotide polymorphisms (SNPs) derived mainly from Bos indicus breeds, and using five reference taurine breeds (n = 212). Population genetic differentiation (FST ) values across sliding windows were estimated between zebu and reference combined taurine breeds. The most differentiated regions (FST  ≥ 0.53), representing the top 1% smoothed FST values, were considered to represent regions under diversifying selection. In total, 285 and 317 genes were identified in the comparisons of Ethiopian cattle with taurine and Asian zebu with taurine respectively. Some of these genes are involved in stress responses/thermo-tolerance and DNA damage repair (HSPA4, HSF1, CMPK1 and EIF2AK4), pigmentation (ERBB3 and MYO1A), reproduction/fertility (UBE2D3, ID3 and PSPC1), immune response (PIK3CD and AKIRIN2) and body stature and size (MBP2, LYN and NPM1). Additionally, the candidate genes were associated with functional terms (e.g. cellular response to stress, DNA repair, inflammatory response) important for physiological adaptation to environmental stresses. The results of our study may shed light on the influence of artificial and natural selection in shaping the genomic diversity of modern cattle breeds and also may serve as a basis for further genetic investigation of traits of tropical adaptation in cattle.

Genome-wide genetic diversity, population structure and admixture analysis in African and Asian cattle breeds.

Knowledge about genetic diversity and population structure is useful for designing effective strategies to improve the production, management and conservation of farm animal genetic resources. Here, we present a comprehensive genome-wide analysis of genetic diversity, population structure and admixture based on 244 animals sampled from 10 cattle populations in Asia and Africa and genotyped for 69,903 autosomal single-nucleotide polymorphisms (SNPs) mainly derived from the indicine breed. Principal component analysis, STRUCTURE and distance analysis from high-density SNP data clearly revealed that the largest genetic difference occurred between the two domestic lineages (taurine and indicine), whereas Ethiopian cattle populations represent a mosaic of the humped zebu and taurine. Estimation of the genetic influence of zebu and taurine revealed that Ethiopian cattle were characterized by considerable levels of introgression from South Asian zebu, whereas Bangladeshi populations shared very low taurine ancestry. The relationships among Ethiopian cattle populations reflect their history of origin and admixture rather than phenotype-based distinctions. The high within-individual genetic variability observed in Ethiopian cattle represents an untapped opportunity for adaptation to changing environments and for implementation of within-breed genetic improvement schemes. Our results provide a basis for future applications of genome-wide SNP data to exploit the unique genetic makeup of indigenous cattle breeds and to facilitate their improvement and conservation.

Linkage disequilibrium and genomic scan to detect selective loci in cattle populations adapted to different ecological conditions in Ethiopia.

Despite the wide range of observed phenotypic diversities and adaptation to different ecological conditions, little has been studied regarding the genetics of adaptation in the genome of indigenous cattle breeds of developing countries. Here, we investigated the linkage disequilibrium (LD) and identified the subset of outlier loci that are highly differentiated among cattle populations adapted to different ecological conditions in Ethiopia. Specifically, we genotyped 47 unrelated animals sampled from high- versus low-altitude environments using a Bovine 50K SNP BeadChip. Linkage disequilibrium was assessed using both D' and r(2) between adjacent SNPs. We calculated FST and heterozygosity at different significance levels as measures of genetic differentiation for each locus between high- and low-altitude populations following the hierarchical island model approach. We identified 816 loci (p < 0.01) showing selection signals and are associated with genes that might have roles in local adaptation. Some of them are associated with candidate genes that are involved in metabolism (ATP2A3, CA2, MYO18B, SIK3, INPP4A, and IREB2), hypoxia response (BDNF, TFRC, and PML) and heat stress (PRKDC, CDK1, and TFDC). Average r(2) and D' values were 0.14 ± 0.21 and 0.57 ± 0.34, respectively, for a minor allele frequency (MAF) ≥ 0.05 and were found to increase with increasing MAF value. The outlier loci identified in the studied Ethiopian cattle populations indicate the presence of genetic variation produced/shaped by adaptation to different environmental conditions and provide a basis for further validation and functional analysis using a reasonable sample size and high-density markers.

Microsatellite DNA markers indicate three genetic lineages in East Asian indigenous goat populations.

The genetic differentiation and phylogenetic relationships of 18 indigenous goat populations from seven East Asian countries were analysed based on data obtained from 26 microsatellite DNA markers. The mean number of alleles (MNA) per population ranged from 2.5 to 7.6, with an average of 5.8. Genetic variability estimated from MNA and heterozygosity (H(E) and H(O) ) were relatively low in coastal and island populations. A heterozygous deficiency within populations (F(IS) = 0.054, P < 0.001) and total inbreeding (F(IT) = 0.181, P < 0.01) were observed, and genetic differentiation in the populations (F(ST) ) was 13.4%. The results of Bayesian model-based clustering and a neighbour-joining tree based on Nei's genetic distance showed that Asian goat populations could be subdivided into at least the following three genetic clusters: East Asian, Southeast Asian and Mongolian. These results are in close accordance with conventional morphological and geographical classifications and migration history.

Variation in mitochondrial DNA and maternal genetic ancestry of Ethiopian cattle populations.

This study describes complete control region sequences of mitochondrial DNA (mtDNA) from 117 Ethiopian cattle from 10 representative populations, in conjunction with the available cattle sequences in GenBank. In total, 79 polymorphic sites were detected, and these defined 81 different haplotypes. The haplotype and nucleotide diversity of Ethiopian cattle did not vary among the populations studied. All mtDNA sequences from Ethiopian cattle converged into one main maternal lineage (T1) that corresponds to African Bos taurus cattle. According to the results of this study, no zebu mtDNA haplotypes have been found in Ethiopia, where the most extensive hybridization took place on the African continent.

Microsatellite analysis reveals high genetic diversity but low genetic structure in Ethiopian indigenous cattle populations.

Ethiopian cattle are under threat from uncontrolled mating practices and are at high risk of becoming genetically homogeneous. Therefore, to evaluate genetic diversity, population structure and degree of admixture, 30 microsatellite markers were genotyped using 351 DNA samples from 10 Ethiopian cattle populations and the Holstein breed. The mean number of alleles per cattle population ranged from 6.93 +/- 2.12 in Sheko to 7.50 +/- 2.35 in Adwa. The mean observed and expected heterozygosities were 0.674 +/- 0.015 and 0.726 +/- 0.019 respectively. Ethiopian cattle populations have maintained a high level of within-population genetic differentiation (98.7%), the remainder being accounted for by differentiation among populations (1.3%). A highly significant deficiency in heterozygotes was detected within populations (F(IS) = 0.071; P < 0.001) and total inbreeding (F(IT) = 0.083; P < 0.001). The study populations were highly admixed but distinct from pure Bos taurus and Bos indicus breeds. The various levels of admixture and high genetic diversity make Ethiopian cattle populations suitable for future genetic improvement and utilization under a wide range of agro-ecologies in Ethiopia.

Haemophagocytic lymphohistiocytosis in X-linked severe combined immunodeficiency.

Haemophagocytic lymphohistiocytosis (HLH) is characterized by destruction of haematopoietic elements, and is associated with a variety of manifestations including immune abnormalities. We describe an infant with HLH who had no evidence of infection or malignancy. He had markedly reduced natural killer (NK) and T-cell numbers and mitogen responses, consistent with severe combined immune deficiency. Western blot and flow cytometry analyses revealed an absence of interleukin (IL)-2 receptor gamma (gamma common) chain expression and a transition (C --> T) at nucleotide 684 in the gamma common gene. This novel case highlights the need for a thorough evaluation of immunological phenotype and genotype in patients with HLH.

Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase, Lyp.

Protein tyrosine phosphatases act in conjunction with protein kinases to regulate the tyrosine phosphorylation events that control cell activation and differentiation. We have isolated a previously undescribed human phosphatase, Lyp, that encodes an intracellular 105-kD protein containing a single tyrosine phosphatase catalytic domain. The noncatalytic domain contains four proline-rich potential SH3 domain binding sites and an NXXY motif that, if phosphorylated, may be recognized by phosphotyrosine binding (PTB) domains. Comparison of the Lyp amino acid sequence with other known proteins shows 70% identity with the murine phosphatase PEP. The human Lyp gene was localized to chromosome 1p13 by fluorescence in situ hybridization analysis. We also identified an alternative spliced form of Lyp RNA, Lyp2. This isoform encodes a smaller 85-kD protein with an alternative C-terminus. The lyp phosphatases are predominantly expressed in lymphoid tissues and cells, with Lyp1 being highly expressed in thymocytes and both mature B and T cells. Increased Lyp1 expression can be induced by activation of resting peripheral T lymphocytes with phytohemagglutinin or anti-CD3. Lyp1 was found to be constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. Overexpression of lyp1 reduces Cbl tyrosine phosphorylation, suggesting that it may be a substrate of the phosphatase. Thus, Lyp may play a role in regulating the function of Cbl and its associated protein kinases.

Cloning and characterization of the human homolog of mouse Jak2.

Members of the Jak family play a critical role in signal transduction mediated by cytokine and hormone receptors. In this study, we report the cloning and characterization of human Jak2. The predicted amino acid sequence shows 91% homology to the described murine Jak2, but with a significant difference in the extreme C-terminal sequence. Using the human cDNA as a probe, we localized the gene for human Jak2 to chromosome 9p23-24. Human Jak2 mRNA is highly expressed in the spleen, lymph nodes, and peripheral blood lymphocytes (PBLs). A polyclonal antibody raised against the unique C-terminus of human Jak2 was used to characterize Jak2 protein. Levels of Jak2 protein expression increased significantly in mitogen- and anti-IgM-stimulated B cells and to a lesser degree in activated T cells. In addition, high levels of Jak2 protein were detected in pre-B leukemia cells.

Jak3 activation in human lymphocyte precursor cells.

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4- CD8-) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) beta gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.

Human immune disorder arising from mutation of the alpha chain of the interleukin-2 receptor.

Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor alpha chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment, 20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines, suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells, and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias, attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker, AG-490, selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death, with no deleterious effect on normal haematopoiesis.

JAK3 protein tyrosine kinase mediates interleukin-7-induced activation of phosphatidylinositol-3' kinase.

The interleukin-7 (IL-7) receptor is expressed throughout T-cell differentiation and, although lacking a tyrosine kinase domain, mediates tyrosine phosphorylation in T cells. We have identified IL-7-induced activation of three cyoplasmic tyrosine kinases in T cells, Jak1, Jak3, and the src-like kinase p56lck. Many members of the cytokine receptor superfamily activate the Jak protein tyrosine kinase family, with resultant phosphorylation of the Stat transcriptional activator factors. We describe here a novel function of the Jak kinases, because Jak kinase activity is not only required for Stat activation but also for P13 kinase response to IL-7 in human T cells. We show that IL-7 receptor-mediated Jak activation can occur independently of p56lck activity. IL-7-induced P13 kinase activation, mediated by tyrosine phosphorylation of the P13 kinase p85 subunit, is essential to the IL-7 proliferative signal and also occurs in the absence of src family kinase activity. Jak3 is found associated with the p85 subunit of P13 kinase in an IL-7-responsive manner in T cells and appears to regulate IL-7-induced P13 kinase activation by mediating tyrosine phosphorylation of the p85 subunit. Specific inhibition of IL-7-induced Jak kinase activity ablates p85 tyrosine phosphorylation, subsequent P13 kinase activation, and, ultimately, proliferation. The ability to regulate P13 kinase activity indicates a more generalized role for the Jak family than activation of gene transcription via the Stat family in cytokine receptor signal transduction.

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity.

Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI-3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.

Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.

We have previously described a type of selective T cell deficiency (STD) characterized by persistent infections reminiscent of severe combined immunodeficiency. We show here that STD patients carry a mutation of zap-70, resulting in loss of the activity of this kinase. The thymi of zap-70-/- patients show the presence of CD4+CD8+ cells in the cortex; however, only CD4, not CD8, single-positive cells are present in the medulla. Peripheral CD4+ T cells from the zap-70-/- patients exhibit markedly reduced tyrosine phosphorylation, fail to produce interleukin-2, and do not proliferate in response to T cell receptor stimulation by mitogens or antigens. Thus, Zap-70 kinase appears to be indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells.

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor on human thymocytes.

Interleukin-7 (IL-7) is a glycoprotein that regulates lymphocyte precursor growth and differentiation. However, the exact mechanism whereby the IL-7 receptor (IL-7R) mediates these cell growth signals remains unknown. One of the earliest metabolic events linked to mitogenic responses in other growth factor receptor systems is the activation of phosphatidylinositol-3 kinase (PI-3 kinase). We demonstrate here that ligation of the IL-7R results in dose- and time-dependent increases in PI-3 kinase activity. These results suggest that PI-3 kinase is involved in signal transduction via the IL-7R in human thymocytes.

Interleukin 7 receptor mediates the activation of phosphatidylinositol-3 kinase in human B-cell precursors.

Interleukin 7 appears to be central to promoting growth and differentiation of lymphocyte precursors. Pro-B cells, as well as pre-pre-B cells, but not mature cells express the IL-7 receptor suggesting an important role for IL-7 during these early stages of B cell maturation. Indeed, IL-7 was shown to promote not only growth but also immunoglobulin gene rearrangement in these cells. However, the mechanism by which IL-7 receptor transduces these growth signals remains elusive. In an attempt to characterize a pathway of signal transduction via the IL-7 receptor, we have demonstrated here that ligation of the IL-7 receptor is associated with increased activity of PI-3 kinase. PI-3 kinase products were detected by TLC and their identity confirmed by HPLC. Further, by in vivo labeling intact cells we have shown that IL-7 receptor mediates the activation of PI-3 kinase.

CD44 in human placenta: localization and binding to hyaluronic acid.

CD44, a receptor for hyaluronic acid (HA), has been identified in the stroma of stem and terminal chorionic villi of human term placenta. The CD44 glycoprotein antigen, isolated from placenta by affinity to monoclonal antibody (mAb) 50B4, consisted mainly of species of M(r) 85,000 and 200,000. Radiolabelled CD44 bound specifically to HA attached to plastic, predominantly via the M(r) 85,000 species; this binding was inhibited by soluble HA and hyaluronidase. The binding of CD44 to HA was also inhibited by mAb 50B4 and IM7.8.1, which recognize epitopes of cluster I and II respectively, but was not blocked by a polyclonal antibody to peptide 18-30 of the B loop (residues 12-101). These results suggest that the portion of the B loop of CD44 implicated in the binding to HA is between amino acids 31-101 and that epitopes located outside the B loop, such as that recognized by mAb IM7.8.1 (between residues 132-215), contribute to this interaction. The presence of a functional CD44 molecule in the human term placenta suggest a role for this molecule in situ in the stabilization and orientation of HA network important in the maintenance of the structural integrity of the placenta.

CD10 and CD44 genes of leukemic cells and malignant cell lines show no evidence of transformation-related alterations.

The expression of CD10/CALLA is associated primarily with childhood leukemia of pre-B lymphocyte phenotype. We have compared the hybridization pattern of the CALLA gene from leukemic and normal cells digested with several restriction enzymes. No alterations were noticed with Eco RI, Sac I, Pvu II, Eco RV, Hind III, and Msp I. Since CALLA is also found on other malignancies, we analyzed DNA samples prepared from cell lines derived from leukemia, lymphoma, glioblastoma, retinoblastoma, and neuroblastoma. Normal restriction patterns were observed for all the lines regardless of their CALLA phenotype. Having demonstrated previously that CALLA was structurally identical to neutral endopeptidase 3.4.24.11 (NEP), we have now established a correlation between surface expression of CALLA and NEP activity on leukemia samples and on several cell lines. Malignant cells tested expressed a functionally active enzyme and no gross alteration was present in the CALLA gene. The CD44 gene is expressed on most cells of hemopoietic origin and on greater than 95% of cases of acute lymphoblastic leukemia and acute myeloblastic leukemia studied. It is also expressed on normal astrocytes and on malignant cells of glioma/astrocytoma types. We now report that a similar pattern of hybridization was observed with Sac I, Pvu II, and Eco RI for leukemic samples, normal cells, and malignant cell lines. A polymorphism was recently detected for CD44 using Hind III; leukemic cells and malignant lines also showed this normal polymorphism. Thus no deletion or insertion could be detected in the CD44 gene of leukemic cells and malignant lines, suggesting that no gross DNA alterations were involved. The correlation between surface expression and enzymatic activity of CD10/CALLA and the expression of CD44 on a variety of malignant cells would suggest that the structure and function of these two gene products are probably not altered by the process of transformation.