Characterization of the human lineage-specific pericentric inversion that distinguishes human chromosome 1 from the homologous chromosomes of the great apes

The human and chimpanzee genomes are distinguishable in terms of ten gross karyotypic differences including nine pericentric inversions and a chromosomal fusion. Seven of these large pericentric inversions are chimpanzee-specific whereas two of them, involving human chromosomes 1 and 18, were fixed in the human lineage after the divergence of humans and chimpanzees. We have performed detailed molecular and computational characterization of the breakpoint regions of the human-specific inversion of chromosome 1. FISH analysis and sequence comparisons together revealed that the pericentromeric region of HSA 1 contains numerous segmental duplications that display a high degree of sequence similarity between both chromosomal arms. Detailed analysis of these regions has allowed us to refine the p-arm breakpoint region to a 154.2 kb interval at 1p11.2 and the q-arm breakpoint region to a 562.6 kb interval at 1q21.1. Both breakpoint regions contain human-specific segmental duplications arranged in inverted orientation. We therefore propose that the pericentric inversion of HSA 1 was mediated by intra-chromosomal non-homologous recombination between these highly homologous segmental duplications that had themselves arisen only recently in the human lineage by duplicative transposition.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Justyna M. Szamalek and Violaine Goidts are contributed equally to the paper.     

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.     

Dual role of the Jak1 FERM and kinase domains in cytokine receptor binding and in stimulation-dependent Jak activation

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-gammaRalpha. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-gammaRalpha when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.     

Enzyme-dependent variations in the polysialylation of the neural cell adhesion molecule (NCAM) in vivo

Polysialic acid (polySia), an alpha2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.     

A high density of X-linked genes for general cognitive ability: a run-away process shaping human evolution?

The incidence of mental disability is 30% higher in males than in females. We have examined entries in the OMIM database that are associated with mental disability and for several other common defects. Our findings indicate that compared with the autosomes, the X chromosome contains a significantly higher number of genes that, when mutated, cause mental impairment. We propose that these genes are involved in the development of cognitive abilities and thus exert a large X-chromosome effect on general intelligence in humans. We discuss these conclusions with regard to the conservation of the vertebrate X-chromosomal linkage group and to human evolution.     

Regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae by inositol and choline: kinetics of repression and derepression

The biosynthesis of phosphatidylserine (PS) and its conversion to phosphatidylcholine (PC) are regulated coordinately by inositol and choline in Saccharomyces cerevisiae (G. M. Carman and S. A. Henry, 1989, Annu. Rev. Biochem. 58, 635-669). In this study, PS decarboxylase activity is shown to be partially repressed when inositol is added to the medium of cells in the log phase of growth, and the extent of repression is augmented by the inclusion of choline, but not ethanolamine. The kinetics of repression and derepression of PS decarboxylase, PS synthase, and phospholipid N-methyltransferase (PNMT) activities, as regulatory responses to the availability of exogenous inositol and choline, have been characterized. When inositol was added to the medium of cell cultures growing exponentially, the three biosynthetic enzyme activities reached an intermediate level of repression (50-85% of control) within 60 min. After the addition of the combination of inositol and choline, PS decarboxylase, PS synthase, and PNMT activities decreased to the intermediate levels of repression in 60 min and were subsequently reduced to 15-40% of control values during a later stage of regulation (2-3 h). In a derepression study, the three enzyme activities remained relatively stable for approximately 60 min following the removal of choline and/or inositol from the growth medium, but the specific activities of PS decarboxylase, PS synthase, and PNMT increased to maximally derepressed levels within 2-3 h. The induction of the three biosynthetic activities was blocked by cycloheximide, but not by chloramphenicol. In summary, the level of PS decarboxylase activity in S. cerevisiae is partially and reversibly suppressed by inositol and further diminished by the combination of inositol and choline. The biphasic kinetics of repression by inositol and choline suggest that the effect of choline is dependent on earlier events mediated by inositol and possibly involves a separate regulatory factor(s).     

Geranylgeraniol Promotes Entry of UT-2 Cells into the Cell Cycle in the Absence of Mevalonate

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5–10 μMGG-OH. Under these growth conditions [³H]GG-OH is actively incorporated into UT-2 proteins. Prominent [³H]geranylgeranylated polypeptides in the size range (19–27 kDa) of the small GTP-binding proteins are observed by SDS–PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [³H]geranylgeranylated cysteine residues. Even though [³H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 μMdid not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.