A molecular comparison of T lymphocyte populations infiltrating the liver and circulating in the blood of patients with chronic hepatitis B: Evidence for antigen-driven selection of a public complementarity-determining region 3 (CDR3) motif

Despite a large number of T cells infiltrating the liver of patients with chronic hepatitis B, little is known about their complexity or specificity. To characterize the composition of these T cells involved with the pathogenesis of chronic hepatitis B (CHB), we have studied the clonality of V beta T cell receptor (TCR)-bearing populations in liver tissue by size spectratyping the complementarity-determining region (CDR3) lengths of TCR transcripts. We have also compared the CDR3 profiles of the lymphocytes infiltrating the liver with those circulating in the blood to see whether identical clonotypes may be detected that would indicate a virus-induced expansion in both compartments. Our studies show that in most of the patients examined, the T cell composition of liver infiltrating lymphocytes is highly restricted, with evidence of clonotypic expansions in 4 to 9 TCR V beta subfamilies. In contrast, the blood compartment contains an average of 1 to 3 expansions. This pattern is seen irrespective of the patient's viral load or degree of liver pathology. Although the TCR repertoire profiles between the 2 compartments are generally distinct, there is evidence of some T cell subsets being equally distributed between the blood and the liver. Finally, we provide evidence for a putative public binding motif within the CDR3 region with the sequence G-X-S, which may be involved with hepatitis B virus recognition.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Searching for missing pieces of the sex-determination puzzle

Little is known of the mechanisms whereby the mammalian indifferent gonad develops into a testis or ovary. In XY individuals, Sry, the mammalian testis-determining gene, is expressed in the pre-Sertoli cells, which then differentiate into Sertoli cells. Other cell types, which include the germ cells, the steroidogenic cells and the connective tissue cells, must then be instructed to develop in a male-specific manner. Although some genes involved in sex-determination and differentiation processes have been identified, we know little of how they interact and cooperate to orchestrate the development of a testis or ovary. We have initiated an expression-screening program designed to identify additional genes, known or novel, which play a role in these processes. This approach is based on our belief that many of the genes we seek will be expressed in a sex-specific manner during the period of sex-determination and differentiation. Most of the genes identified previously are transcription factors and so we aim, in particular, to find genes involved in cell-to-cell communication, signal transduction, and transcriptional regulation, downstream of the differentiation of Sertoli cells. We have used a suppression subtractive-hybridization method to generate male- and female-enriched probes and libraries. Clones are validated as being sex-specific in their expression patterns by array screening and in situ hybridization. Here we report on our progress to date and the general applicability of the approach for studies in other systems. J. Exp. Zool. 290:517-522, 2001. (C) 2001 Wiley-Liss, Inc.

Spatially dynamic expression of Sry in mouse genital ridges

We have studied the spatial dynamics of Sry transcription in the genital ridges of mouse embryos. We find that Sry is expressed in a dynamic wave that emanates from the central and/or anterior regions, extends subsequently to both poles, and ends in the caudal pole. This dynamism may explain the relative positioning of ovarian and testicular tissue seen in ovotestes in mice. Since direct regulatory targets of SRY ought to be expressed in a corresponding or complimentary wave, our observations pave the way for identification of target genes. Sry is expressed in internal cells but not in coelomic surface epithelial cells, indicating that its effect on proliferation of surface cells is achieved non-cell-autonomously. The cellular dynamism of Sry expression revealed in this study thus provides important insights into both the cellular and molecular mode of action of SRY, and how perturbations in Sry expression can lead to anomalies of sexual development. (C) 2001 Wiley-Liss, Inc.

Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) beta-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class 11 antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-gamma) in periodontitis patients, whereas P. gingivalis GroEL did not induce any, skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-gamma or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type I cytokine profile in their peripheral blood T-cell pools.

Charting the course of ovarian development in vertebrates

The decision of the embryonic gonad to differentiate as either a testis or an ovary is a critical step in vertebrate development. The molecular basis of this decision has been the focus of much study, particularly over the past decade. Here we contrast the knowledge of early gonadal development and the switch to testis differentiation with the lack of molecular understanding of ovarian development at early stages. We review current knowledge regarding mechanisms of ovarian morphogenesis and propose a model for the hierarchical control of development of the fetal ovary, incorporating the few genes already known to be important and several signals or factors that are hypothesised to exist in the early ovary.

Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development

Blepharophimosis ptosis epicanthus inversus syndrome (BPES) is a human disorder caused by mutations in the forkhead transcription factor gene FOXL2 and is characterized by facial dysmorphology combined in some cases with ovarian failure. To better understand the role of FOXL2 in the etiology of ovarian failure in BPES, we examined its expression in embryonic ovaries of mice, chickens, and red-eared slider turtles, representatives of three phylogenetically distant vertebrate groups that have different mechanisms of sex determination. Expression of Foxl2 was detected in early ovaries of all three species around the time of sex determination and was associated with both somatic and germ cell populations in mice. Expression was sexually dimorphic in all cases. Sequence analysis of turtle and chicken FoxL2 orthologues indicated an unusually high degree of structural conservation during evolution. FoxL2 was found to be autosomal in chickens, and therefore unlikely to represent the dominant ovarian-determining gene that has been postulated to exist as a possible explanation for female heterogamety in birds. Our observations suggest that BPES may result from early abnormalities in regulating the development of the fetal ovary, rather than premature degeneration of the postnatal or adult ovary. Further, our results suggest that FOXL2 is a highly conserved early regulator of vertebrate ovarian development.

The ins and outs of transcriptional control: nucleocytoplasmic shuttling in development and disease

Recent findings relating to SOX transcription factors indicate that defects in organogenesis can be caused not only by impairment of the biochemical properties of transcription factors but also, in some cases, by deficient nuclear import. In addition, experimentally interfering with the nuclear export signals of some SOX factors has now been found to cause developmental defects. Controlling the balance of nuclear import and export might be a common means by which transcription factor activity can be regulated during development, and defects in these processes might underlie a broader spectrum of inherited developmental disorders.

Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y-DOM sex reversal

The phenomenon of B6-Y-DOM sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (136) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y-DOM sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y-DOM sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway. (C) 2004 Elsevier Inc. All rights reserved.

Regulation of Amh during sex determination in chickens: Sox gene expression in male and female gonads

During mammalian sexual development, the SOX9 transcription factor up-regulates expression of the gene encoding anti-Mullerian hormone (AMH), but in chickens, Sox9 gene expression reportedly occurs after the onset of Amh expression. Here, we examined expression of the related gene Sox8 in chicken embryonic gonads during the sex-determining period. We found that cSox8 is expressed at similar levels in both sexes at embryonic day 6 and 7, and only at the anterior tip of the gonad, suggesting that SOX8 is not responsible for the sex-specific increase in cAmh gene expression at these stages. We also found that several other chicken Sox genes (cSox3, cSox4 and cSox11) are expressed in embryonic gonads, but at similar levels in both sexes. Our data suggest that the molecular mechanisms involved in the regulation of Amh genes of mouse and chicken are not conserved, despite similar patterns of Amh expression in both species.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

The production of male of-only offspring in beef cattle - a proof of principle project

The Australian beef industry places the greatest value in bulls, in comparison to cows, for prime beef production. Male carcasses can be sold for a larger profit due to their increased muscle mass. This project aims to demonstrate the feasibility of producing male animals that can sire male only offspring, through a transgenic approach in mice that could later be translated into livestock production systems. The mouse Sry (Sex determining region on the Y) gene has been shown to provide the initiating molecular signal leading to male sex determination in mammals. Sry has also been shown to cause sex reversal in XX mice transgenic for the gene. In this project Sry will be targeted to a locus not subject to X-inactivation on the X chromosome of XY mice. These mice will be bred to determine how the transgene is passed on, to determine expression of the transgene, and to assess its activity in causing XX sex reversal. The male mice transgenic for the Sry gene on their X chromosome will be produced using tetraploid aggregation, which in a single step produces 100% ES cell derived embryos. The same target locus can later be used to introduce the bovine SRY gene onto the X chromosome of bovidae species and using germ cell transplantation produce sex reversed animals. This would bypass the need for expensive chimera crosses and provide farmers with a stud bull capable of producing only sons.

Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

Lentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced throughout the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields world wide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include: susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum; Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust, caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to several species of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistance in the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerant germplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this end progress has been made in mapping the lentil genome and several genetic maps are available that eventually will lead to the development of a consensus map for lentil. Marker density has been limited in the published genetic maps and there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic maps and efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for disease resistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, is underway in Australia and Canada and promising results have been obtained. Comparative genomics and synteny analyses with closely related legumes promises to further advance the knowledge of the lentil genome and provide lentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic tools such as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that may eventually be available for use in lentil crop improvement.