Transcervical collection of bovine embryos up to Day 21: an 8-year overview.

Transcervical embryo collection is used routinely in the bovine species throughout the world to collect Day 6 to Day 9 embryos (early embryos) for genetic selection. For research purposes, however, the collection of embryos at later stages of pregnancy, i.e., Days 12 to 21 (late embryos), is needed. So far, for the recovery of late embryos, females are euthanized and embryo collection is performed after recovery of the genital tract. To reduce the number of animals used and still provide valuable material for embryo research, we have therefore developed a transcervical technique to collect late embryos. The objective of this study was to compare embryo recovery results at early and late stages within our laboratory. Altogether, 232 cows were used for this study. One hundred forty-five flushes were performed to collect embryos from Days 6 to 9, and 251 flushes were performed to collect embryos from Days 12 to 21. For the early embryos, a classical three-way collection equipment was used. To collect the late embryos, the same equipment was used, but the extensible flexible catheter that goes inside the external rigid catheter was removed, so that larger embryos could be collected through the remaining larger hole (two-way collection). All females were submitted to ovum pick up to remove the dominant follicle and were subsequently superovulated with FSH. Luteolysis was induced 48 hours before artificial insemination. Two artificial inseminations were performed with frozen semen, 48 and 56 hours after PGF2α injection. Before embryo collection, cows were treated with an epidural injection of a local anesthetic drug. The presence of CL was checked, and they were counted by rectal palpation. For all collections, the cervix was prepared with the initial introduction of a dilator. Then, the catheter was introduced in one horn, and the cuff was inflated as low as possible. For the collection of late embryos, the flushing solution (30 mL) was injected slowly twice to suspend the embryos before flushing the horn with 500 mL, and the same operation was performed on the second horn. There was no significant difference in the number of embryos collected per flush in the early- and late-stage (758 embryos collected, 5.22 ± 6.02 per flush vs. 1238 embryos collected, 4.93 ± 5.07 per flush, respectively). The number of embryos collected per CL, however, was significantly lower in the early versus late group (0.39 ± 0.32% vs. 0.44 ± 0.34%, respectively). The late collection allowed the retrieval of full conceptuses (embryonic and extraembryonic tissues), even at very late stages such as Days 18 to 21. Careful collection is needed, however, so that conceptuses are not damaged or torn: the horn must be massaged gently and the flush should be ideally recovered in one single flow. This technique is a powerful tool to collect the late-stage embryos for research purposes. Because it is not traumatic, animals can be used again for the same procedure.

Spatial and temporal changes of decorin, type I collagen and fibronectin expression in normal and clone bovine placenta.

INTRODUCTION: Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after somatic cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies. METHODS: Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and somatic cloning pregnancies. RESULTS: The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta. DISCUSSION: In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes. CONCLUSIONS: These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

Energy and lipid metabolism gene expression of D18 embryos in dairy cows is related to dam physiological status.

We analyzed the change in gene expression related to dam physiological status in day (D)18 embryos from growing heifers (GH), early lactating cows (ELC), and late lactating cows (LLC). Dam energy metabolism was characterized by measurement of circulating concentrations of insulin, glucose, IGF-1, nonesterified fatty acids, ß-hydroxybutyrate, and urea before embryo flush. The metabolic parameters were related to differential gene expression in the extraembryonic tissues by correlation analysis. Embryo development estimated by measuring the length of the conceptuses and the proportion of expected D18 gastrulating stages was not different between the three groups of females. However, embryo metabolism was greatly affected by dam physiological status when we compared GH with ELC and GH with LLC but to a lesser extent when ELC was compared with LLC. Genes involved in glucose, pyruvate, and acetate utilization were upregulated in GH vs. ELC conceptuses (e.g., SLC2A1, PC, ACSS2, ACSS3). This was also true for the pentose pathway ( PGD, TKT), which is involved in synthesis of ribose precursors of RNA and DNA. The pathways involved in lipid synthesis were also upregulated in GH vs. ELC. Despite similar morphological development, the molecular characteristics of the heifers' embryos were consistently different from those of the cows. Most of these differences were strongly related to metabolic/hormone patterns before insemination and during conceptus free-life. Many biosynthetic pathways appeared to be more active in heifer embryos than in cow embryos, and consequently they seemed to be healthier, and this may be more conducive to continue development.

Gene selection heuristic algorithm for nutrigenomics studies.

Large datasets from -omics studies need to be deeply investigated. The aim of this paper is to provide a new method (LEM method) for the search of transcriptome and metabolome connections. The heuristic algorithm here described extends the classical canonical correlation analysis (CCA) to a high number of variables (without regularization) and combines well-conditioning and fast-computing in "R." Reduced CCA models are summarized in PageRank matrices, the product of which gives a stochastic matrix that resumes the self-avoiding walk covered by the algorithm. Then, a homogeneous Markov process applied to this stochastic matrix converges the probabilities of interconnection between genes, providing a selection of disjointed subsets of genes. This is an alternative to regularized generalized CCA for the determination of blocks within the structure matrix. Each gene subset is thus linked to the whole metabolic or clinical dataset that represents the biological phenotype of interest. Moreover, this selection process reaches the aim of biologists who often need small sets of genes for further validation or extended phenotyping. The algorithm is shown to work efficiently on three published datasets, resulting in meaningfully broadened gene networks.

Pre- and post-partum mild underfeeding influences gene expression in the reproductive tract of cyclic dairy cows.

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post-partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post-partum patterns of IGF-1, insulin, ß-hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid-related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post-partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post-partum dairy cows.

Expression and localization of DLX3, PPARG and SP1 in bovine trophoblast during binucleated cell differentiation.

The differentiation of the trophoblast is marked in ruminants by the formation of binucleated cells (BNC). They appear from pre-implantation onwards but the molecular mechanisms underlying their differentiation remain largely unexplored. Taking advantage of our recent data, we analyzed the expression pattern of DLX3 and PPARG that are known regulators of early placenta formation and extended our analysis to one of their potential regulators, SP1. Our study is the first to demonstrate the co-expression of DLX3, PPARG and SP1 in bovine BNC nuclei. This suggests a possible role of these transcription factors through BNC specific genes at the time of pre-placental differentiation.

Identification of lactic acid bacteria involved in the spoilage of pasteurized "foie gras" products.

The spoiling microflora of a re-packaged French "foie gras" product was studied. A total of 54 isolates, originating from two different factories, were identified using phenotypical and molecular methods (partial 16S rDNA sequencing). Weissella viridescens was the main species detected in the products from factory 1 (64% of the isolates). These products had a low lactic acid concentration and were considered as non-spoiled. The microflora of factory 2 was dominated mainly by the genus Lactobacillus (95% of the isolates), and the high lactic acid concentration of these products was linked with a strong spoilage. Among the 30 Lactobacillus strains, three species were predominant: Lactobacillus sakei (nine isolates), Lactobacillus coryniformis (eight isolates) and Lactobacillus paraplantarum (five isolates). Challenge tests were performed to confirm the involvement of the Lactobacillus strains in the spoilage of the product. Sterile "foie gras" samples were inoculated with 14 LAB strains from the collection. The most acidifying strains belonged to the species L. sakei, Lactobacillus plantarum and L. paraplantarum. This confirmed the role of the strains from the Lactobacillus genus as the main spoilers of "foie gras" products and will be useful to design new quality protocols and extend the shelf-life of these products.

Abnormal expression of the imprinted gene Phlda2 in cloned bovine placenta.

Cloning in mammals suffers from high rates of pregnancy losses associated with abnormal placentation, mainly placentomegaly, leading to fetal death. Placental growth is dependent on the regulated expression of many genes of which imprinted genes play a fundamental role. Among them, the Phlda2 gene is expressed from the maternal allele and acts to limit placental growth in mouse and human. Here we used Northern blots, quantitative RT-PCR and in situ hybridization to analyze the expression patterns of bovine PHLDA2 and to compare its expression levels in normal and somatic cell nuclear transfer (SCNT) placentas over a range of gestational stages. PHLDA2 is not expressed in extra-embryonic tissues before d32 of gestation but the level of expression increases throughout pregnancy until term in the placental villi collected from pregnancy obtained by artificial insemination (AI). At all stages of pregnancy, PHLDA2 mRNA are specifically localized in the trophoblast mononucleated cells contrasting with lack of expression in the binucleated cells and uterine tissues. In SCNT placentas, a similar pattern of expression was observed during early pregnancy. In contrast the level of expression is significantly reduced around d200 of gestation in the placental villi from pathological clones. The reduced expression of PHLDA2 was obvious particularly in the placental villi anchored within the uterine crypts with expression confined to the trophoblast of the chorionic plate. Altogether, these results highlight a similarity in expression patterns for PHLDA2 bovine and human where expression is localized to the trophoblast throughout pregnancy and parallels the continuous growth of the placenta. Moreover, the lack of expression in the fetal villi from oversized bovine cloned placenta is consistent with the function of PHLDA2 in restraining placental growth and underlines an aberrant expression of this gene after somatic cloning.

Embryonic stem cells in cattle.

Because of the potential use of embryonic stem cells (ESC), especially for genetic modifications, there is great interest in establishing domestic animals-related ESCs. Unfortunately, despite considerable efforts, validated ESC lines in species other than mice and primates are yet to be isolated. In this paper, we will summarize the current knowledge on bovine ESCs in an attempt to understand why derivation of domestic animal ESC is still unsuccessful and we will discuss some promising future approaches.

Placental expression of major histocompatibility complex class I in bovine somatic clones.

Abnormally increased placental expression of major histocompatibility complex class I (MHC-I) molecules at the trophoblastic surface has been suggested previously to be the cause of early fetal loss in nuclear transfer (NT) bovine pregnancies. Here, we report the lack of expression of MHC-I at the trophoblastic surface at D30 and D60 and in placentomes from D60 to term in placentas obtained by NT from three different genotypes and by artificial insemination, whatever the outcome of the pregnancy. MHC-I expression was assessed by immunohistochemistry using four different antibodies, including a novel beta2-microglobulin antibody. The MHC-I type of the clones was established using reference strand-mediated conformation analysis (RSCA); however, since it proved problematic to type the recipient animals in the same way, outcome of pregnancy could not be related to MHC compatibility. In conclusion, the present study provides no evidence to support abnormal expression of MHC-I on the trophoblastic surface in clones as a major cause of fetal loss during pregnancy after NT.

Gene expression in elongating and gastrulating embryos from ruminants.

In ruminants, more than 30% of the embryonic losses observed after artificial insemination (AI) have an early origin, coincident with a marked elongation of the trophoblast which occurs before implantation. Several observations provide clear evidence that early elongation of the conceptus relies on cell multiplication, cell growth and cell shape remodeling. Recent results indicating an intense multiplication of a non-fully differentiated trophoblast, which still expresses some epiblast genes, has to be considered at the onset of elongation. It has also been shown in the last two years that general metabolism and protein trafficking are characteristic of the onset of elongation whereas cellular interactions, cell to cell signaling and cell adhesion become predominant at the end of elongation. Accordingly, expression of most of the single genes identified so far increases during elongation and is related to the establishment of embryo-maternal exchanges before implantation. However, not much is known of what controls the induction of the elongation process or the coordinated development of the embryonic and extra-embryonic tissues. This review highlights new information on this developmental phase and summarizes the views on the complex cross-talk among molecules which might govern conceptus development and lead to successful implantation.

A cDNA macroarray resource for gene expression profiling in ruminant tissues involved in reproduction and production (milk and beef) traits.

cDNA arrays have proven to be useful tools to screen gene expression in many animal species including livestock species. A collaborative program was launched to construct a ruminant cDNA collection, representative of three tissues: Muscle, Embryo and Mammary gland, named MEM. This collection gathers clones mainly arising from 3 non-normalised cDNA libraries: a directed bovine muscle library, a 14-day-old bovine embryo library and a goat lactating mammary library. It is made up of 1896 clones (637 muscle, 882 embryo and 377 mammary cDNAs), selected after sequencing and bioinformatic analyses. Amplification products yielded from these clones as well as controls were printed onto Nylon membranes to generate macroarrays. Hybridisation with relevant cDNA targets allowed checking the location of about 50 cDNAs and the specificity of each sub-set of the repertoire. Macroarrays were hybridised with radiolabelled cDNA complex targets from five different tissues (muscle, embryo, mammary gland, adipose tissue and oocyte). Both somatic and germinal complex targets gave valid hybridisation signals with 45 to 80% of the printed probes. This specific cDNA collection now provides a powerful tool for transcriptomic studies with the ultimate objective to better understand physiological and metabolic functions in ruminants. It will be subsequently included into a forthcoming larger collection.

Nuclear transfer technologies: between successes and doubts.

Cloning of mammals by nuclear transfer can lead to the birth of healthy adult animals but more often compromises the development of the reconstructed embryos. A high incidence of fetal and postnatal losses has been observed in several species, revealing the existence of long-lasting effects induced by the nuclear transfer procedures. Remodeling of donor chromatin by the recipient cytoplasm after nuclear transfer is frequently associated with the deregulation of specific genes, and recent observations point to the potential importance of time-dependent DNA methylation events in the occurrence of these alterations. Screening strategies to design nuclear transfer procedures that would mimic the epigenetic remodeling occurring in normal embryos are being designed, and improvement in the efficiency of procedures could imply a pre-conditioning of donor cells. Early mammalian development appears to be rather tolerant to epigenetic abnormalities, raising the possibility that even a fully functional reprogrammed genome may have been subjected to some epigenetic alterations. Bringing nuclear transfer to routine practice requires greater knowledge and understanding of the basic biological processes underlying epigenetic controls of nuclear activities. An important issue at present is to limit the production of those aberrant phenotypes that may result in significant insult to the nature and welfare of animals.

Brachyury is expressed in gastrulating bovine embryos well ahead of implantation.

Brachyury is a T-box-containing transcription factor involved in mesoderm formation during vertebrate gastrulation. To analyse whether the regulation of gastrulation varies as much as the timing of gastrulation does with respect to implantation, we isolated a bovine brachyury cDNA fragment. The amino acid sequence shows high similarity to mouse and human Brachyury and clear differences to other T-box genes. Whole-mount in situ hybridisation reveals a normal expression pattern except for a transiently reduced expression in the anterior part of the primitive streak. According to these results, gastrulation in mammals is implemented and regulated irrespective of implantation.

Subtractive hybridization used to identify mRNA associated with the maturation of bovine oocytes.

The main objective of this project is to identify mRNA associated with oocyte maturation and embryonic developmental competency. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryo. We used bovine slaughterhouse-recovered ovaries and collected the oocytes from two follicle size categories: <2 mm and 3-5 mm. The mRNA content of oocytes from follicles 3-5 mm where considered to be more competent when compared to the content of oocytes from follicles <2 mm. In this report we compare two different technical approaches both involving PCR to compare the mRNA pools of the oocytes. In the first approach we performed the differential display (DDRT) technique to amplify and display side by side the cDNAs of groups of 10 denuded oocytes. From this approach, we isolated 28 different bands. After analysis, three of those bands had strong homology with known genes. In the second approach pools of 50 denuded oocytes were submitted to suppressive subtraction hybridization (SSH). We identified several known genes like cyclin B1, splicing factor ccl.4, cytochrome c oxidase, and mineralocorticoid receptor while numerous other clones remain unidentified. The cyclin B1 clone was used as a probe to evaluate its follicular size specificity on virtual Northern blot. The PCR basis of these techniques allows comparison of mRNA from tissues of low abundance such as oocytes. In this study the SSH resulted in longer clones than DDRT and showed high specificity.

p34cdc2 expression and meiotic competence in growing goat oocytes.

The expression of the catalytic subunit of the maturation promoting factor (MPF), p34cdc2, was analyzed during meiosis and final growth of goat oocytes. Western blot analysis revealed the presence of p34cdc2 in fully grown oocytes (follicles > 3 mm in diameter) prior to and during meiotic maturation. p34cdc2 was present in partially competent oocytes at the germinal vesicle stage (follicles 0.5 to 0.8 mm and 1 to 1.8 mm in diameter). In contrast, p34cdc2 was not expressed in meiotically incompetent oocytes from small antral follicles (follicles < 0.5 mm in diameter). The amount of p34cdc2 increased with oocyte growth and acquistion of meiotic competence. Moreover, p34cdc2 accumulated in partially competent and incompetent oocytes within 27 hr of culture, but the level of p34cdc2 in incompetent oocytes remained very low and was not sufficient to allow spontaneous resumption of meiosis. The expression of the cdc2 gene was analyzed by polymerase-chain-reaction (PCR) on reverse transcribed mRNA. The presence of the cdc2 transcript was evidenced in both competent and incompetent oocytes at the germinal vesicle stage. These data indicate that a deficiency in the expression of p34cdc2 that could be regulated at the translational level, may be a limiting factor for meiotic competence acquistion in goat oocytes. We further investigated the mechanisms of MPF activation in competent and incompetent oocytes by using okadaic acid, a protein phosphatase inhibitor. Okadaic acid induced the premature resumption of meiosis associated with MPF activation in competent oocytes. In partially competent oocytes, okadaic acid induced premature meiosis reinitiation and MPF activation, but only after pre-culture for 10 hr. Acquisition of sensitivity to okadaic acid treatment was dependent on protein synthesis since it failed to occur when cycloheximide was added during the pre-culture period. Incompetent oocytes responded to okadaic acid treatment only after 27 hr of culture, when they had accumulated small amounts of p34cdc2. These data suggest that okadaic acid may bypass the subthreshold level of p34cdc2, provided the oocytes have synthesized some additional factors that remain to be identified.

Cyclin B1 expression in meiotically competent and incompetent goat oocytes.

To start determining the nature of meiotic incompetence in goat oocytes, we have examined the expression of one of the potential pre-MPF subunits: the cyclin B1. We have been isolating a small DNA probe encoding the goat cyclin B1 box to analyze the expression of the cyclin B1 gene in competent and incompetent goat oocytes. This probe was easily obtained by polymerase-chain-reaction (PCR) on reverse-transcribed mRNA from granulosa cells, using cyclin B specific primers derived from a bovine cDNA. The transcript corresponding to cyclin B1 in goat granulosa cells is 1.8 kb. In situ hybridization analysis indicated that competent and incompetent oocytes contained cyclin B1 mRNA, but also that active cyclin B1 mRNA synthesis occurred at the end of the growth phase, e.g., when oocytes progressed in the acquisition of meoitic competence. Western blot analysis, performed with a monoclonal anticyclin B1 antibody, revealed in competent and incompetent oocytes a polypeptide of 65 kDa corresponding to the goat cyclin B1 protein. This pattern of cyclin B1 expression further suggested that meiotic incompetence in goat oocytes could not be primarily correlated with a lack of cyclin B1 protein as potential pre-MPF subunit, but to a limiting amount of this protein.

GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis.

GLOBOSA (GLO) is a homeotic gene whose mutants show sepaloid petals and carpelloid stamens. The similarity of Glo mutants to those of the DEFICIENS (DEFA) gene suggests that the two genes have comparable functions in floral morphogenesis. The GLO cDNA has been cloned by virtue of its homology to the MADS-box, a conserved DNA-binding domain also contained in the DEFA gene. We have determined the structure of the wild type GLO gene as well as of several glo mutant alleles which contain transposable element insertions responsible for somatic and germinal instability of Glo mutants. Analyses of the temporal and spatial expression patterns of the DEFA and GLO genes during development of wild type flowers and in flowers of various stable and unstable defA and glo alleles indicate independent induction of DEFA and GLO transcription. In contrast, organ-specific up-regulation of the two genes in petals and stamens depends on expression of both DEFA and GLO. In vitro DNA-binding studies were used to demonstrate that the DEFA and GLO proteins specifically bind, as a heterodimer, to motifs in the promoters of both genes. A model is presented which proposes both combinatorial and cross-regulatory interactions between the DEFA and GLO genes during petal and stamen organogenesis in the second and third whorls of the flower. The function of the two genes controlling determinate growth of the floral meristem is also discussed.

Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development.

We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis-acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non-permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.

Genomic organization of the mouse T cell receptor V alpha family.

Based on the analysis of V alpha gene segment deletions in a panel of T lymphomas, we have constructed a map of the mouse T cell receptor alpha/delta region and assigned the relative position of 72 distinct V gene segments. Three major observations have emerged from such studies. First, members of a given V alpha subfamily are not organized in discrete units along the chromosome but largely interspersed with members of other V alpha subfamilies. Second, analysis of the deletion map suggests the existence of repetitive patterns (V alpha clusters) in the chromosomal distribution of the V alpha gene segments. Third, the present-day organization of the V alpha/delta region may be readily explained by a series of sequential duplications involving three ancestral V alpha clusters. Direct evidence for the existence of these unique structural features has been gained by cloning approximately 370 kb of DNA and positioning 26 distinct V alpha gene segments belonging to six different subfamilies. Finally, the relationships existing between the V alpha/delta gene segment organization and usage are discussed in terms of position-dependent models.