Showing papers by "Magdalena Skipper published in 2002"

A different exchange-rate mechanism

Abstract: Meiosis-specific processes, such as pairing of homologous chromosomes and recombination, are highly conserved, nevertheless there have been conflicting theories about the sequence of events that leads up to recombination. In yeast, it is well established that meiotic exchange can take place in the absence of the synaptonemal complex (SC)  a proteinaceous ‘glue’ that holds sister chromatids together. However, in a recent issue of Genes and Development, Page and Hawley provide new evidence that, in flies, the SC is essential for the initiation of recombination, and that there might be some unexpected and fundamental differences between meiosis in flies and in yeast. Previous evidence from Drosophila mutants has shown that the relationship between recombination and the SC is not the same as it is in yeast. For example, the mei-W68 mutant has a normal SC but does not undergo recombination and the c(3)G mutant eliminates both the SC and recombination. To investigate whether recombination depends on the SC, Page and Hawley have cloned c(3)G and shown that it encodes an essential component of the SC. As expected, C(3)G localizes between the paired homologues, and this localization is altered in mutants that disrupt the SC, confirming its role as an integral part of the SC. Using C(3)G as a marker, Page and Hawley looked at the SC in mutants in which meiotic exchange is defective. One class of these mutants has reduced frequency of exchange, and the distribution of crossover sites is biased against distal parts of the chromosomes  a phenomenon known as polar effect. Because C(3)G is mislocalized in these mutants, the authors conclude that incorrect SC assembly might, at least partially, account for the polar effect. But even in normal cells, crossovers are not randomly distributed along the chromosome because of crossover interference  the suppression of crossovers in the immediate neighbourhood of an established crossover point. In yeast, incorrect assembly of the SC and incomplete pairing of the homologues abolishes interference, but in Drosophila, Page and Hawley show that when the SC is absent because C(3)G is mislocalized or non-functional, interference is essentially unaffected. The picture that emerges is that, in contrast to yeast, which might use the initiation of recombination to align homologous chromosomes, and the SC just to stabilize their pairing, Drosophila needs the SC for the initial alignment of the homologues, without which recombination will not be initiated. It also seems that these two organisms have different ways of controlling interference  in yeast this process is SC dependent, whereas in flies it is SC independent. It remains to be seen which way is more common among other organisms. Magdalena Skipper

Tracking positive selection

Abstract: It takes two, so the song goes, and for genes, this is often so. Recessive disorders, for example, require both copies of a gene to be lost. However, some diseases and traits can occur when one wild-type allele is still present. Now, two papers report that a BLM gene mutation — which, when homozygous, causes the recessive, cancer-predisposition disease Bloom syndrome (BS) — surprisingly predisposes mice and humans to intestinal cancer when haploinsufficient. Because BLM is a helicase that maintains genome stability, these papers highlight the crucial role of genome instability in both cancer pathogenesis and predisposition. Heppner Goss et al. generated a new BS mouse model by targeting the mouse Blm gene with a mutation (Blm) that causes a premature truncation. This mutation acts as a null allele and simulates a founder mutation (BLM) that is present in 1% of Ashkenazi Jews. The authors used two approaches to investigate the effect of Blm haploinsufficiency on tumorigenesis in these mice. First, they injected them with murine leukaemia virus (MLV). Both wild-type and Blm mice developed metastatic T-cell lymphoma on exposure to this virus, but mutant mice died earlier, despite tumour morphology being the same in both sets of mice. Next, they crossed Blm mice to Apc min mice — a mouse model of familial adenomatous polyposis coli. (The min mutant was chosen because the gastrointestinal (GI) tract is where cancer commonly develops in BS patients.) Double heterozygous mutant mice developed twice as many GI adenomas as did Apc /Blm animals, and many Apcmin/Blm+/− mice had tumours with high-grade dysplasia. Tumours in both Apc/Blm and Apcmin/Blm+/− also showed Apc loss of heterozygosity (LOH). In both mutants, Apc LOH seemed to occur predominantly through the loss of chromosome 18, where Apc is located. However, in some Apcmin/Blm+/− tumours, Apc loss also occurred through somatic

Developmental biology: The making of an organ

Abstract: Update on GM On 4 February 2002, the Royal Society released a report entitled ‘Genetically modified plants for food use and human health  an update’. Prepared by a group of experts, led by Jim Smith from the Wellcome/CRC in Cambridge, UK, it focused on “the effects that GM foods might have on human health” (The Royal Society report). It states that “the dangers of eating GM food were negligible” (Financial Times), and that “GM products licensed for sale in Britain are safe” (The Independent). The report questions “whether commonly used safety tests for modified food are adequate” (The Independent) and calls for more rigorous tests. At present, GM safety assessment relies on a principle of “substantial equivalence”, “whereby a new GM crop is deemed safe if it is essentially the same as the unmodified equivalent” (Financial Times). But the experts found this method insufficient, and warned that genetic modification “could lead to unpredicted harmful changes in the nutritional state of foods” (The Guardian). There was special concern about the potential effects of GM food on those with restricted nutritional intake, such as babies, and “... the poor of Central America, who have maize as 50% of their food ... [and] might be affected by poorer nutritional standards in the new crops” (The Guardian). The experts also called “for more work to rule out possible links between GM crops and the development of allergic reactions” (The Independent). The report coincides with a finding that “super weeds ... in field margins or some distance from GM crops” acquired “genes from modified crops and ... became resistant to a series of herbicides” (The Guardian). Magdalena Skipper IN THE NEWS

Gene Finding: Seek and you shall find...

Abstract: Genome biologists dream of defining a full complement of genes in a given genome, but gene prediction is notoriously difficult. Now, Kumar et al. describe a global strategy for gene finding that combines expression and homology data, with which they have discovered new Saccharomyces cerevisiae genes, and which is likely to be widely applicable. Since the completion of the yeast genome sequence in 1996, ~60 new genes have been identified mainly from expression or homology data. Kumar et al. used these two information sources to carry out a global search of the yeast genome for previously unidentified genes. Their analysis began with a gene trap in which a promoterless, 5′ truncated lacZ reporter incorporated within a Tn3 transposon was randomly inserted throughout the yeast genome. The resulting gene fusions were then detected in a highthroughput screen for β-galactosidase activity, and the trapped ORFs were identified by sequencing the insertion junctions. Previously unannotated ORFs identified in this way were studied further only if they were at least 25 codons long, in an intergenic region or orientated antisense to previously annotated genes. Their expression was then assessed by microarray analysis, in which labelled poly(A) RNA was hybridized to a specifically designed oligonucleotide array that carries sense and antisense sequence from the newly discovered ORFs. Only the ORFs that showed strong expression in these strand-specific assays were considered to be bona fide genes. This experimental approach was also complemented with a computational analysis, in which Kumar et al. searched the yeast genome against metazoan and prokaryotic genome data and against the SWISS-PROT database. Out of the total 137 new genes that Kumar et al. identified, 104 were less than 100 codons long  because short genes are notoriously difficult to predict, this is a reflection of the strength of this strategy for gene identification. The authors also uncovered a whole new family of subtelomerically located genes and augmented a class of previously poorly represented genes that overlap the already annotated genes but lie antisense to them.

Developmental biology: Becoming competent

Abstract: The key functions of the pancreas are to regulate digestion and sugar metabolism. Our desire to understand the development of this organ is driven not only by the need to understand organ formation, but also by the hope that we can recapitulate it in vitro, thus creating a supply of insulin-producing cells for diabetes therapy. An important step in this direction is now provided by Kawaguchi and colleagues, who report that the pancreatic transcription factor Ptf1a is required in mice to commit cells to the pancreatic fate and for their subsequent proliferation and differentiation. Ptf1a was previously implicated in the development of the exocrine pancreas — the portion that is responsible for secreting digestive enzymes. Little was known about Ptf1a-expressing cells during early pancreatic development, so the authors did careful recombinationbased lineage tracing in mice in which the expression of a lacZ reporter was activated by the endogenous Ptf1a promoter in normal and in Ptf1a-deficient mice. Because the activated lacZ allele is expressed independently of cell fate, the progeny of cells in which the Ptf1a promoter had been activated could be definitively identified. In the normal pancreas, Kawaguchi et al. found that cells from both the exocrine and the endocrine — hormone-producing — pancreas express Ptf1a early in their lineage history. Most importantly, they saw that Ptf1a deficiency causes large numbers of lacZexpressing cells to appear in the intestinal epithelium. So, the presence or absence of Ptf1a seems to be crucial in determining how endodermal progenitors choose between organ fates. As another test of whether Ptf1a is expressed in all pancreatic precursors, the authors used its promoter to drive the expression of Pdx1 — a gene that is essential for pancreas formation. It turned out that Ptf1a-driven expression of Pdx1 was sufficient to restore the formation of all pancreatic

Human genetics: Sticking together

Abstract: Nephronophthisis is the most common genetic cause of chronic renal failure in children. Mutations in three different loci contribute to this recessive phenotype, one of which, NPHP1, encodes nephrocystin — a novel docking protein that is involved in cell adhesion. Now, Mollet et al. and Otto et al. report the characterization of a fourth locus, NPHP4, that contributes to this disorder. Mollet et al. also show that its product interacts with nephrocystin, probably functioning in the same pathway. Because of genetic heterogeneity of nephronophthisis, Mollet et al. and Otto et al. embarked on finding new loci that are linked with this disorder. To this end, both groups used genome-wide linkage and haplotype analysis in families in which there was no linkage between the disorder and the previously identified loci. The results implicated a small region on chromosome 1 that contained six candidate genes, so both groups screened affected individuals for mutations in a subset of candidates that were known to be expressed in the kidney. Collectively, the two groups found 16 mutations in one ORF that encodes a novel hydrophilic protein. Mollet et al. call this protein nephrocystin-4, whereas Otto et al. call it nephroretinin, to reflect the fact that the mutations in NPHP4 are found in some individuals who not only suffer from nephronophthisis but also retinitis pigmentosa. The product of NPHP4 has been conserved during evolution — the mouse orthologue is 86% identical with the human protein at the amino-acid level, and there is also a previously uncharacterized worm orthologue. Although novel, the NPHP4 protein contains a prolinerich region with a consensus motif that is known to interact with SH3 domains, one of which is present in nephrocystin. Mollet et al. showed that NPHP4 interacts with nephrocystin, at least in vitro. But domains other than SH3 must also be involved in this interaction because it was not abolished by a mutation that disrupts the SH3 domain of nephrocystin. Given that nephrocystin interacts with several proteins that are involved in cell adhesion, the authors speculate that the product of NPHP4 also affects the same process and that pathogenic changes that are associated with nephronophthisis result from the abnormal adhesion of cells in the renal tubules. The results of both studies indicate the existence of a new cell-adhesion pathway that is important in the nephronophthisis disease process. But the pathway remains to be investigated in detail. The fact that Otto et al. failed to detect mutations in NPHP4 in some of the affected families indicates a greater genetic heterogeneity than expected for nephronophthisis that awaits additional studies. Magdalena Skipper

Functional genomics: Fully functional

Abstract: LEONARD I. ZON CHILDREN’S HOSPITAL, BOSTON, USA The ultimate goal of functional genomics is to determine the function of every gene in a given organism. To this end, systematic projects have begun to knock out all of the genes in some organisms whose genomes have been sequenced. Saccharomyces cerevisiae has led the way, and Giaever et al. now report in Nature the creation and initial functional analysis of strains that, collectively, are deleted for 96% of this yeast’s genes. This pioneering analysis substantially enhances our knowledge of yeast biology. Using mitotic recombination, the authors deleted the complete coding sequence of 96% of all annotated yeast ORFs. The function of each deleted gene was assayed in several growth conditions, and, because of the unique 20-nucleotide molecular barcode included in the deletion cassette, all deletion strains could be analysed in parallel. The more important a gene was for growth under a given condition, the quicker the strain in which it had been deleted diminished in culture, so that genes could be ranked in the order of their importance for growth under a specific condition. The authors tested the fitness of their deletion strains on media that contained different amino acids and carbon sources, when osmolarity, pH and salinity were altered, and in the presence of an antifungal agent. In each case, new genes were identified. For example, ten new genes were found to be involved in growth on galactose. This was quite a surprise, given that galactose metabolism is among the best-studied pathways in yeast. Another surprising finding was that the expression of many genes that were important for growth in a given medium didn’t change under these conditions; conversely, expression of those that were not required, did change. Fitness profiling of a collection of yeast deletion strains was also carried out by Steinmetz et al. who focused on mitochondrial genes. Having identified 466 genes that are important for mitochondrial function, they used genomic map positions to identify human orthologues that are linked with heritable diseases. These results have important implications for expression profiling studies that have not been confirmed by functional data, as they indicate that fitness profiling can refine expression profiling data. This approach provides systematic, unbiased information about gene function and marks the beginning of a fully functional era of genomics. It will make those who work on other organisms for which full genome sequences are available pursue their deletion projects even more zealously. Magdalena Skipper

A balancing act

Abstract: LEONARD I. ZON CHILDREN’S HOSPITAL, BOSTON, USA Heart development requires a delicate balance of proliferation and differentiation that is underpinned by the finely tuned execution of genetic programmes. Chen et al. and Shin et al. now identify and characterize a new mouse homeobox gene, Hop, which, although unable to bind DNA, modulates cardiac-specific gene expression by interacting with known major players in cardiogenesis. Both groups isolated Hop from a mouse EST database while searching for new homeobox genes expressed in the heart. Sequence and experimental analysis of Hop’s homeodomain revealed that this 73 amino-acid protein is unlikely to have retained its DNA binding ability. Not surprisingly, Hop is expressed in the heart. Its expression in the embryonic heart starts shortly after that of Nkx2-5 — a principal activator of heart-specific transcription — suggesting that it might be one of its targets. The fact that Hop expression is downregulated in Nkx2-5-null mice confirmed this prediction. Chen et al. also found Nkx2-5 consensus binding sites upstream of Hop and showed that Nkx2-5 binds them in vitro, again confirming that Hop is a direct target of Nkx2-5. Approximately half of the Hopnull homozygotes generated by both groups have abnormal myocardium and die during embryogenesis from heart failure. Shin et al. found that Hop−/− embryos that died had thin, often ruptured ventricle walls, whereas Hop−/− adults had thickened ventricle walls, mainly as a result of increased numbers of cardiomyocytes. The authors explain this paradoxical observation by evoking a dual role for Hop in heart development — first it acts to expand the myocardium, whereas later it restricts its proliferation. Hop seems to fulfil its functions by modulating a subset of heartspecific genes. Although it cannot bind directly to their promoters, it can affect their expression by binding to another crucial transcription regulator in the developing heart, Srf (serum response factor), therefore preventing it from activating genes downstream. Both groups showed that Hop — which might be vertebrate specific — seems to be involved in a balance between myocardial expansion and restriction that is necessary for proper heart development. We now know that it achieves this by modulating the action of Srf, but what of its other targets? Shin et al. have already made the first step towards addressing this question by using microarray analysis to look at the differences in gene expression between Hop−/− and wild-type mice. Among many future directions of research is Hop’s involvement in heart disease — understanding Hop function might be relevant to continuing efforts to regrow heart muscle by manipulating the transition of myoblasts from proliferation to terminal differentiation. Magdalena Skipper

Gene expression: Peripheral oscillations

Abstract: NATURE REVIEWS | GENETICS VOLUME 3 | JUNE 2002 | 411 Circadian rhythms are most often studied in association with behaviour and the underlying gene-expression changes in the brain. But circadian clocks that generate transcription periodicity also exist in many peripheral organs, as discussed in a new study published in Nature that demonstrates extensive circadian gene regulation in the hearts and livers of mice. In the biggest study of its kind, the authors show that this type of gene regulation is more extensive than previously thought and that, although it has a specialized role in a given tissue, it also has a broad biological function. Although the fact that peripheral tissues have circadian regulation is not new, the extent of this regulation remained largely unknown. To investigate this, Storch et al. performed a microarray analysis on the hearts and livers of mice whose light/dark cycle had been synchronized. Using a set of genes with known circadian regulation as a guide, they found that 10% of liver genes and 8% of heart genes were regulated by the circadian clock. Although oscillations in gene expression were generally out of phase with each other in the liver, they were much more synchronized in the heart. Interestingly, there was very little overlap between the genes that oscillate in the heart and in the liver — only 37 genes shared the same phase of peak expression. The authors point out that the core set of genes is important as it is likely to include those that are important for the functions of the peripheral clocks. Among them, they found genes involved in chromatin regulation, such as DNMT1-associated protein-1, and in ubiquitin pathways, suggesting that gene expression, in general, and protein stability are influenced by circadian rhythms. To determine the kinds of processes that involve genes with circadian regulation, Storch and colleagues used a gene-ontology approach to sort the data into three categories: biological process, molecular function and cellular component. Their analysis indicated that, even in a single tissue, the circadian clock regulates many diverse processes, several of which are shared by the liver and the heart. So, the authors conclude that, although the genes regulated by circadian clocks are different in the two tissues, this type of regulation influences many related or overlapping processes. As the authors point out, there are some limitations to their study. They admit that transcript oscillations need not be reflected at the protein level, which depends, at least in part, on protein turnover. Also, for some proteins, circadian oscillations might have little physiological consequence. Nonetheless, together with another study published in Cell, which looked at circadian gene regulation in the liver and in a part of the hypothalamus that is known to harbour the ‘pacemaker’ of circadian control, this study substantially improves our understanding of circadian gene expression and provides evidence about the core clock mechanism that underlies it. Magdalena Skipper