Drivers of tree fecundity in pedunculate oak (Quercus robur) refugial populations at the species' southwestern range margin.

The current low latitudinal range margins of many extra-tropical plant species consist of small and scattered populations that persist locally in microrefugia. It remains poorly understood how their refugial distribution affects mating patterns and reproductive success. Here we examine flower and acorn production and their determinants in refugial populations of the widespread European forest tree pedunculate oak (Quercus robur). We monitored male flower, female flower and acorn production in 159 adult trees from 12 oak stands over 2 years. We related these and derived parameters to a series of ecological and genetic predictor variables extrinsic (stand size, density and isolation as well as elevation, topography and forest cover) or intrinsic (size, phenology and several genotypic measures) to the target tree. Tree fertility was unrelated to extrinsic factors but determined by tree size, although we detected size-independent variation in reproductive investment. Female flower number accurately predicted acorn crop size. Fruit set differed between years, evidencing the existence of pollen limitation at the landscape but not at the local scale. Fruit set also tended to increase with the number of mates of the target tree. We detected no other evidence for genetic constraints on mating. Reproduction was triggered by a combination of small-scale and landscape-scale drivers. Although short-distance mating prevailed, limited pollen flow did not appear to significantly constrain reproductive success. The high intrinsic ability of populations to maintain their reproductive capacity may help explain their successful long-term persistence in an adverse broader environment.

Unusually limited pollen dispersal and connectivity of Pedunculate oak (Quercus robur) refugial populations at the species' southern range margin.

Low-latitudinal range margins of temperate and boreal plant species typically consist of scattered populations that persist locally in microrefugia. It remains poorly understood how their refugial habitats affect patterns of gene flow and connectivity, key components for their long-term viability and evolution. We examine landscape-scale patterns of historical and contemporary gene flow in refugial populations of the widespread European forest tree Pedunculate oak (Quercus robur) at the species' southwestern range margin. We sampled all adult trees (n = 135) growing in a 20 km long valley and genotyped 724 acorns from 72 mother trees at 17 microsatellite loci. The ten oak stands that we identified were highly differentiated and formed four distinct genetic clusters, despite sporadic historical dispersal being detectable. By far most contemporary pollination occurred within stands, either between local mates (85.6%) or through selfing (6.8%). Pollen exchange between stands (2.6%) was remarkably rare given their relative proximity and was complemented by long-distance pollen immigration (4.4%) and hybridization with the locally abundant Quercus pyrenaica (0.6%). The frequency of between-stand mating events decreased with increasing size and spatial isolation of stands. Overall, our results reveal outstandingly little long-distance gene flow for a wind-pollinated tree species. We argue that the distinct landscape characteristics of oaks' refugial habitats, with a combination of a rugged topography, dense vegetation and humid microclimate, are likely to increase plant survival but to hamper effective long-distance pollen dispersal. Moreover, local mating might be favoured by high tree compatibility resulting from genetic purging in these long-term relict populations.

Isolation and characterization of 16 polymorphic microsatellite loci for Frangula alnus (Rhamnaceae).

We report the first 16 polymorphic nuclear microsatellite markers developed for Frangula alnus (Rhamnaceae). Markers were tested on all three subspecies as well as on three local populations, including analyses of both leaf and seed endocarps. A total of 87 alleles were found (mean number of alleles per locus was 5.44) for 72 individuals genotyped. Observed and expected heterozygosities ranged from 0.097 to 0.792 and from 0.093 to 0.794, respectively. The levels of polymorphism and exclusionary power of the developed markers render them applicable for parentage analyses and measurements of seed dispersal through direct comparison of endocarps and adult tree genotypes.

Isolation and characterization of 12 microsatellite loci for Rhamnus alaternus (Rhamnaceae).

Twelve polymorphic microsatellite loci were developed for Rhamnus alaternus using an enriched-library approach. We detected 69 alleles in 49 individuals genotyped (mean number of alleles per locus was 4.79) in two different populations. The values of observed and expected heterozygosities ranged from 0.045 to 0.963 and 0.089 to 0.873 respectively. Levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for studies of contemporary pollen and seed gene flow through parentage analyses.

Rangewide phylogeography of a bird-dispersed Eurasian shrub: contrasting Mediterranean and temperate glacial refugia.

We studied the phylogeography of alder buckthorn (Frangula alnus), a bird-dispersed shrub or small tree distributed over most of Europe and West Asia and present in three of the four main refugia of West Palaearctic temperate woody plants: the Iberian Peninsula, the Balkans and Anatolia. A total of 78 populations from 21 countries were analysed for chloroplast DNA variation using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and 21 different haplotypes were distinguished. We found a very strong overall population differentiation (GST = 0.81) and phylogeographical structure, and a sharp contrast between the haplotype-rich refugia and the almost completely uniform area of postglacial colonization. The haplotype network comprises three lineages made up of haplotypes from the Iberian Peninsula, Anatolia with the Caucasus, and temperate Europe. The Iberian and the Anatolian branches represent parts of a major lineage that spans over the whole northern Mediterranean Basin and some neighbouring areas and probably dates back to the Tertiary. Many haplotypes of this lineage are distributed locally and most populations are fixed for a single haplotype; these populations have apparently been very stable since their establishment, experiencing negligible gene flow and few mutations. The temperate European lineage consists of one very widespread and abundant plus six locally distributed haplotypes. Four of them are located in Southeast Europe, the putative refugium of all extant temperate European populations. Contrary to populations from Iberia and Anatolia, F. alnus populations from the southeastern European refugium have most genetic variation within populations. Bird-mediated seed dispersal has apparently allowed not only a very rapid postglacial expansion of F. alnus but also subsequent regular seed exchanges between populations of the largely continuous species range in temperate Europe. In contrast, the disjunct F. alnus populations persisting in Mediterranean mountain ranges seem to have experienced little gene flow and have therefore accumulated a high degree of differentiation, even at short distances. Populations from the southern parts of the glacial refugia have contributed little to the postglacial recolonization of Europe, but their long-term historical continuity has allowed them to maintain a unique store of genetic variation.

Transcriptional analysis of the 69-kb sequence centromeric to HLA-J: a dense and complex structure of five genes.

Performed within the framework of the sequencing of the 356-kb MHC class I distal region, systematic bioinformatic annotation and preliminary experiments conducted on the whole sequence indicate a high level and a complex pattern of expression. In this paper, we analyze a particular stretch of 69 kb centromeric to the HLA-J gene, in which we identify 21 different mRNAs mainly expressed in testis, and characterize five different transcription units, HZFw, HZFc, HCGV, HTEX6, and HTEX4. These tightly linked genes form a cluster conserved between human and mouse and displaying a high gene density of about one every 14 kb. Alternative splicing processes are observed for all the genes, together with an alternative polyadenylation event for gene HTEX4, sense/antisense mRNA overlaps for HZFw and HZFc, for HZFw and HCGV at their 3' end, and for HTEX6 and HTEX4 at their 5' end. This complex genomic structure suggests a mechanism of coregulation by cis-interaction in gene expression.

A 356-Kb sequence of the subtelomeric part of the MHC Class I region.

The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3. The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence. Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases. Most of the ESTs are clustered in two regions. In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes.

Sequence analysis of two genomic regions containing the KIT and the FMS receptor tyrosine kinase genes.

The KIT and FMS tyrosine kinase receptors, which are implicated in the control of cell growth and differentiation, stem through duplications from a common ancestor. We have conducted a detailed structural analysis of the two loci containing the KIT and FMS genes. The sequence of the approximately 90-kb KIT locus reveals the position and size of the 21 introns and of the 5' regulatory region of the KIT gene. The introns and the 3'-untranslated parts of KIT and FMS have been analyzed in parallel. Comparison of the two sequences shows that, while introns of both genes have extensively diverged in size and sequence, this divergence is, at least in part, due to intron expansion through internal duplications, as suggested by the discrete extant analogies. Repetitive elements as well as exon predictions obtained using the GRAIL and GENEFINDER programs are described in detail. These programs led us to identify a novel gene, designated SMF, immediately downstream of FMS, in the opposite orientation. This finding emphasizes the gene-rich characteristic of this genomic region.

Systematic sequencing of the human HLA-A/HLA-F region: establishment of a cosmid contig and identification of a new gene cluster within 37 kb of sequence.

The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization. To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing. This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids. Four new genes, organized with the HCG-V gene in a clustered structure, have been identified. Two of these contain a zinc finger motif characteristic of DNA-binding proteins. The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region. The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase IA12.2 subunit and of the murine tctex6 gene. Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene. This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.

The 9E3/CEF4 cytokine: kinetics of secretion, processing by plasmin, and interaction with extracellular matrix.

The chicken gene, 9E3/CEF4, is a small inducible cytokine highly homologous to human IL-8 and gro alpha. It is overexpressed during wound healing and in the tissues around tumours induced by Rous sarcoma virus. More is known about the expression of 9E3 in vivo than any other of the small cytokines, yet little is known about its biochemical characteristics and functions. Here we report on some of the biochemical properties of the 9E3 gene product, the kinetics of protein secretion, the post-secretory processing of the protein, and on its association with ECM molecules. The protein: (1) is synthesized and secreted in < 10 min; (2) is not glycosylated and does not bind heparin with high affinity; (3) is secreted as a 9 kDa form and is processed to a 6-7 kDa form by plasmin, an enzyme released at wound sites and produced in association with tumours; (4) the small form binds to interstitial collagen, laminin and to a lesser extent to proteoglycan, and does not bind to collagen IV or fibronectin. This is the most rapidly secreted protein yet described in eukaryotic cells and is the first of the small inducible cytokines to be found to associate with ECM molecules other than glycosaminoglycans. Our results suggest that, given the appropriate stimulus, the level of the 9E3 cytokine could be elevated very rapidly, resulting in similarly rapid biological responses. The different modes of availability of the two forms of the molecule suggest that the two isoforms may play different roles in vivo.

A new non-HLA multigene family associated with the PERB11 family within the MHC class I region.

In an effort to initiate steps designed to characterize the idiopathic hemochromatosis disease gene, the HLA-A/HLA-F region where this gene is in disequilibrium linkage with some polymorphic markers has been overlapped by a yeast artificial chromosome (YAC) contig. In order to achieve the physical mapping of these YACs and of the corresponding genomic region, we subcloned one of the YACs involved. A computer-assisted analysis of the sequence of one subclone led to the isolation of a potential exon that proved to belong to a new expressed messenger named HCGIX. After Southern blot analysis, the corresponding cDNA clone was found to belong to a new multigene family whose members are dispersed throughout the HLA class I region and are closely associated with members of another recently described multigene family designated PERB11. The data reported here suggest that these two multigene families form a cluster that have been dispersed together throughout the telomeric part of the major histocompatibility complex and have been involved in the genesis of this human class I region.

Structure and function of the long terminal repeat of the chimpanzee foamy virus isolates (SFV-6).

The complete long terminal repeat (LTR) nucleotide sequence of the chimpanzee foamy virus isolate SFV-6 was determined. Its 1761-bp size makes it the longest LTR reported to date among all retroviruses. Since the length of its LTR is similar to that of other simian isolates while its sequence homology is closer to that of HFV, SFV-6 genetic structure appears to be intermediate between simian and human foamy viruses. Transient expression assays demonstrate that SFV-6 encodes a transactivator of viral gene expression directed either by its own LTR or by heterologous promoters like HFV and HIV-1 LTRs. Our data also provide evidence for cross-transactivation between SFV-6 and HFV.

Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells.

9E3/CEF4, which is released from transformed chicken embryo fibroblasts (CEF), is a member of the platelet factor 4 family of inflammatory proteins and may be the avian homolog of interleukin-8. Since the function of 9E3/CEF4 is unknown, we examined the effect of the protein on mitogenicity and chemotaxis, as well as its expression, in fibroblasts and peripheral blood cells. 9E3/CEF4 mRNA was expressed in chicken peripheral blood monocytes, and its expression was stimulated by incubation of the monocytes with lipopolysaccharide or phorbol myristic acetate. Boyden double-membrane analysis of chemotaxis showed that 9E3/CEF4 was chemotactic for chicken peripheral blood mononuclear cells, as well as for heterophils. Untransformed CEF and CEF transformed with Rous sarcoma virus also migrated to 9E3/CEF4 protein, as measured by Boyden single-membrane analysis. 9E3/CEF4 was slightly mitogenic for CEF, causing a doubling of [3H]thymidine uptake when added to serum-starved CEF.9E3/CEF4 was found associated not only with the cell and in the culture medium of Rous sarcoma virus-transformed CEF but also with the extracellular matrix. The in vivo role of 9E3/CEF4 may be involved with chemotaxis and metastasis, rather than with direct stimulation of mitogenicity.

Small deletion in v-src SH3 domain of a transformation defective mutant of Rous sarcoma virus restores wild type transforming properties.

RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.

Nucleotide sequence of HIV1-NDK: a highly cytopathic strain of the human immunodeficiency virus.

A highly cytopathic strain of HIV1, named HIV1-NDK, has been isolated from a Zaïrian patient affected with AIDS. This isolate is 10(4) times more cytopathic and infectious than the prototype. To correlate the high cytopathic properties of this strain with genetic variations, we have cloned and sequenced the genome of this isolate. The principal feature which could be drawn from the fine analysis of the HIV1-NDK sequence is that the variability is not clustered in one particular region but rather spread out all along the genome. Only minor differences seem to be responsible for the acute biological effect of HIV1-NDK.

Nucleotide sequence and structural organization of the human FMS proto-oncogene.

The human proto-oncogene c-fms [FMS] on chromosome 5q33.3 encodes a transmembrane glycoprotein with tyrosine kinase activity that functions as the cell surface receptor for the macrophage colony stimulating factor (CSF-1 or M-CSF). Overlapping bacteriophage clones that included 35 kb of the FMS locus and contained the complete coding sequence of the CSF-1 receptor were subjected to nucleotide sequencing analysis. Comparison with the cDNA sequence of the human c-fms gene indicated that at least one 5' noncoding exon is located far upstream (ca. 26 kb) from sequences encoding the CSF-1 receptor. The FMS coding sequence consists of 21 small exons and heterogeneously sized introns, ranging from 6.3 kb to less than 0.1 kb in complexity.

78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation.

To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60v-src in the absence of glucose deprivation.

Molecular mechanisms of a t(8;14)(q24;q11) translocation juxtaposing c-myc and TcR-alpha genes in a T-cell leukaemia: involvement of a V alpha internal heptamer.

We have recently described a t(8;14)(q24;q11) translocation which appeared secondarily in a non-established acute T-cell leukaemia and involved the 3' region of c-myc proto-oncogene and the alpha chain of the T-cell receptor gene (TcR-alpha). In order to elucidate the mechanism of the translocation, we have isolated breakpoint regions from normal and recombinant chromosomes. Our results show that the translocation occurred at the 3' end of a V alpha segment in the proximity of recombination signal sequences (5'-heptamer-23bp spacer-nonamer-3'). Interestingly, an inverted heptamer internal to the V alpha segment was found at two nucleotides 5' of the break. Nucleotide sequence analysis also revealed the presence of homologous signal sequences on chromosome 8, suggesting that the recombination enzymatic system played an important role in the generation of the translocation. This hypothesis is supported by the addition of N nucleotides and the loss of only three nucleotides during the rejoining process. These data established the involvement of a V alpha segment and its recombination signals in the mechanisms of t(8;14) translocations in T-cell leukaemias.