Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.

Signaling of need, sibling competition, and the cost of honesty

Young birds and mammals frequently solicit food by means of extravagant and apparently costly begging displays. Much attention has been devoted to the idea that these displays are honest signals of need, and that their apparent cost serves to maintain their honesty. Recent analyses, however, have shown that the cost needed to maintain a fully informative, honest signal may often be so great that both offspring (signaler) and parent (receiver) would do better to refrain from communication. This apparently calls into question the relevance of the costly signaling hypothesis. Here, I show that this argument overlooks the impact of sibling competition. When multiple signalers must compete for the attention of a receiver (as is commonly the case in parent–offspring interactions), I show that (all other things being equal) individual equilibrium signal costs will typically be lower. The greater the number of competitors, the smaller the mean cost, though the maximum level of signal intensity employed by very needy signalers may actually increase with the number of competitors. At the same time, costs become increasingly sensitive to relatedness among signalers as opposed to relatedness between signalers and receivers. As a result of these trends, signaling proves profitable for signalers under a much wider range of conditions when there is competition (though it is still likely to be unprofitable for receivers).

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

A complete genome screen for genes predisposing to severe bipolar disorder in two Costa Rican pedigrees

Bipolar mood disorder (BP) is a debilitating syndrome characterized by episodes of mania and depression. We designed a multistage study to detect all major loci predisposing to severe BP (termed BP-I) in two pedigrees drawn from the Central Valley of Costa Rica, where the population is largely descended from a few founders in the 16th–18th centuries. We considered only individuals with BP-I as affected and screened the genome for linkage with 473 microsatellite markers. We used a model for linkage analysis that incorporated a high phenocopy rate and a conservative estimate of penetrance. Our goal in this study was not to establish definitive linkage but rather to detect all regions possibly harboring major genes for BP-I in these pedigrees. To facilitate this aim, we evaluated the degree to which markers that were informative in our data set provided coverage of each genome region; we estimate that at least 94% of the genome has been covered, at a predesignated threshold determined through prior linkage simulation analyses. We report here the results of our genome screen for BP-I loci and indicate several regions that merit further study, including segments in 18q, 18p, and 11p, in which suggestive lod scores were observed for two or more contiguous markers. Isolated lod scores that exceeded our thresholds in one or both families also occurred on chromosomes 1, 2, 3, 4, 5, 7, 13, 15, 16, and 17. Interesting regions highlighted in this genome screen will be followed up using linkage disequilibrium (LD) methods.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution

Chromosome painting in placental mammalians illustrates that genome evolution is marked by chromosomal synteny conservation and that the association of chromosomes 3 and 21 may be the largest widely conserved syntenic block known for mammals. We studied intrachromosomal rearrangements of the syntenic block 3/21 by using probes derived from chromosomal subregions with a resolution of up to 10–15 Mbp. We demonstrate that the rearrangements visualized by chromosome painting, mostly translocations, are only a fraction of the actual chromosomal changes that have occurred during evolution. The ancestral segment order for both primates and carnivores is still found in some species in both orders. From the ancestral primate/carnivore condition an inversion is needed to derive the pig homolog, and a fission of chromosome 21 and a pericentric inversion is needed to derive the Bornean orangutan condition. Two overlapping inversions in the chromosome 3 homolog then would lead to the chromosome form found in humans and African apes. This reconstruction of the origin of human chromosome 3 contrasts with the generally accepted scenario derived from chromosome banding in which it was proposed that only one pericentric inversion was needed. From the ancestral form for Old World primates (now found in the Bornean orangutan) a pericentric inversion and centromere shift leads to the chromosome ancestral for all Old World monkeys. Intrachromosomal rearrangements, as shown here, make up a set of potentially plentiful and informative markers that can be used for phylogenetic reconstruction and a more refined comparative mapping of the genome.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Effects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancer

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that plays a key role in the differentiation of adipocytes. Activation of this receptor in liposarcomas and breast and colon cancer cells also induces cell growth inhibition and differentiation. In the present study, we show that PPARγ is expressed in human prostate adenocarcinomas and cell lines derived from these tumors. Activation of this receptor with specific ligands exerts an inhibitory effect on the growth of prostate cancer cell lines. Further, we show that prostate cancer and cell lines do not have intragenic mutations in the PPARγ gene, although 40% of the informative tumors have hemizygous deletions of this gene. Based on our preclinical data, we conducted a phase II clinical study in patients with advanced prostate cancer using troglitazone, a PPARγ ligand used for the treatment of type 2 diabetes. Forty-one men with histologically confirmed prostate cancer and no symptomatic metastatic disease were treated orally with troglitazone. An unexpectedly high incidence of prolonged stabilization of prostate-specific antigen was seen in patients treated with troglitazone. In addition, one patient had a dramatic decrease in serum prostate-specific antigen to nearly undetectable levels. These data suggest that PPARγ may serve as a biological modifier in human prostate cancer and its therapeutic potential in this disease should be further investigated.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes

Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecBB allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22–23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-β (TGF-β) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecBB/FecBB ewes were less responsive than granulosa cells from FecB+/FecB+ ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecBB/FecBB ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles.

Potentials of the National Corn Genome Initiative

The present paper summarizes future needs in information and tools, technology, infrastructure, training, funding, and bioinformatics, to provide the genomic knowledge and tools for breeding and biotechnological goals in maize. The National Corn Genome Initiative (NCGA) has developed through actions taken by the National Corn Growers Association (NCGA) and participation in a planning process by institutions, companies, and organizations. At the web address for the NCGI, http://www.inverizon.com/ncgi, are detailed analyses of goals and costs, impact and value, and strategy and approaches. The NCGI has also produced an informative and perceptive video suitable for public groups or schools, about agricultural contributions to life and the place of maize in these contributions. High potential can be expected, from cross-application of knowledge obtained in maize and other cereals. Development of information and tools for all crops, whether monocots or dicots, will be gained through an initiative, and each crop will be positioned to advance with cost-effective parallels, especially for expressed sequences, markers, and physical mapping.

On the origin of and phylogenetic relationships among living amphibians

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.

Retroposon analysis of major cetacean lineages: The monophyly of toothed whales and the paraphyly of river dolphins

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28–33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

Island colonization and evolution of the insular woody habit in Echium L. (Boraginaceae).

Numerous island-inhabiting species of predominantly herbaceous angiosperm genera are woody shrubs or trees. Such "insular woodiness" is strongly manifested in the genus Echium, in which the continental species of circummediterranean distribution are herbaceous, whereas endemic species of islands along the Atlantic coast of north Africa are woody perennial shrubs. The history of 37 Echium species was traced with 70 kb of noncoding DNA determined from both chloroplast and nuclear genomes. In all, 239 polymorphic positions with 137 informative sites, in addition to 27 informative indels, were found. Island-dwelling Echium species are shown to descend from herbaceous continental ancestors via a single island colonization event that occurred < 20 million years ago. Founding colonization appears to have taken place on the Canary Islands, from which the Madeira and Cape Verde archipelagos were invaded. Colonization of island habitats correlates with a recent origin of perennial woodiness from herbaceous habit and was furthermore accompanied by intense speciation, which brought forth remarkable diversity of forms among contemporary island endemics. We argue that the origin of insular woodiness involved response to counter-selection of inbreeding depression in founding island colonies.