An analysis of frequencies of chromosome configurations in wheat and wheat hybrids

Presynaptic association of homologous chromosomes is a prerequisite to the sequence of events that lead to chiasma formation. This association of homologous chromosomes, as entire units, occurs with probability a, and chiasma formation occurs independently in opposite chromosome arms with probability c. a and c have been estimated from frequencies of different chromosome configurations at metaphase I of euhexaploid wheat and several derived lines. In the euploid, a is essentially unity and c is of the order of 95%. All changes in the aneuploidy studied involved c rather than a, whereas the change induced by colchicine application primarily involved a.—Observed and expected frequencies of configurations were compared in wheat hybrids in which only homoeologues were present. The expected frequencies of configurations were estimated from the data, based on a being unity for entire groups of homoeologues and c being the probability of chiasma formation between random homoeologous arms. Observed and expected frequences of configurations were in general agreement; however, an excess of observed closed bivalents at the expense of multivalents is interpreted to mean that not all homoeologues are effectively associated in all cells.—In euhexaploid wheat, we suggest that homologues associate with almost certainty, whereas homoeologous pairs of chromosomes associate less efficiently. The aneuploidy examined in this study does not appear to affect the association of chromosomes, but rather the number of chiasmata that eventuate and, in the case of deficiency of chromosome 5B, the distribution of chiasmata within homoelogues, perhaps by way of rendering sites for chiasma formation of homoelogues more similar.     

Affinity Distance Values among Somatic Metaphase Chromosomes in Maize

In maize root-tip metaphase preparations, all distances between two chromosomes were measured in 50 cells from each of seven stocks and in 30 from one stock; four were arrested with cold, two with 8-hydroxyquinoline, one with colchicine and one with monobromonaphthalene. Standardized, affinity-distance values were calculated for all pairs of homologues and pairs of nonhomologues from each preparation. The homologues of pair X were the least separated, those of pair I the most separated in the cold-arrested stocks. All but pairs I and VIII were shown to be significantly different from the observed mean. The observed mean was less than but not significantly different from the theoretical value for a random distribution. The use of chemical agents for metaphase arrest increased the separation of homologues, except for pair I.—Eleven percent of the comparisons of nonhomologues from cold-arrested, as contrasted to none of the comparisons from the c-metaphase treatments, were significantly different from the theoretical value for a random distribution. This was considered evidence for limited primary nonhomologue association in maize. Although there were specific, differential responses to the two arrest agents, the population of homologous pairs approached a random distribution only in chemically arrested stocks.—Primary homologue association was considered to be maintained by two mechanisms, the more common involving the microtubules and the second involving the nucleolus.—Interpretations are offered regarding the claims of somatic association in other species, especially man. The opportunity in maize for experimentally modifying distance values by cytogenetic techniques is discussed.     

The absence of the somatic association of centromeres of homologous chromosomes in grass mitotic metaphases

Root-tip metaphases from Hordeum vulgare (19 cells), H. marinum (11 cells), Aegilops umbellulata (10 cells) and Zea mays (10 cells) were completely reconstructed from electron micrographs of serially sectioned nuclei. The identity of each chromosome was found by measuring the volumes of its two arms and the presence or absence of a secondary constriction at the nucleolar organising region. With the position of the centromere in three dimensions, these data were used to analyse the relative positions of homologous and heterologous centromeres. In 31 out of the 50 cells analysed, homologues were on average further apart than heterologues. Except for two nucleolar organising chromosomes, there was no evidence of any tendency for the distances between different homologue types to be differently distributed from distances between heterologues. Average distances between homologues of the single nucleolar organising chromosome (linkage group 6) of Zea (2n = 20) were lower than the average for heterologues and the interhomologue distances were distributed significantly differently from the separation distances of chromosome 6 to other chromosomes. Presumably this association occurred because of nucleolar fusion in the previous interphase. Homologues of one of the two nucleolar organising chromosomes of A. umbellulata were also distributed significantly differently from heterologues, with a tendency for homologues to lie farther apart than the average heterologous pair. These results do not support previous work using squashed and spread metaphase preparations (some including abnormal, marked chromosomes) for these species.     

Retrotransposon insertions in rice gene pairs associated with reduced conservation of gene pairs in grass genomes

Small-scale changes in gene order and orientation are common in plant genomes, even across relatively short evolutionary distances. We investigated the association of retrotransposons in and near rice gene pairs with gene pair conservation, inversion, rearrangement, and deletion in sorghum, maize, and Brachypodium. Copia and Gypsy LTR-retrotransposon insertions were found to be primarily associated with reduced frequency of gene pair conservation and an increase in both gene pair rearrangement and gene deletions. SINEs are associated with gene pair rearrangement, while LINEs are associated with gene deletions. Despite being more frequently associated with retrotransposons than convergent and tandem pairs, divergent gene pairs showed the least effects from that association. In contrast, convergent pairs were least frequently associated with retrotransposons yet showed the greatest effects. Insertions between genes were associated with the greatest effects on gene pair arrangement, while insertions flanking gene pairs had significant effects only on divergent pairs.     

An ordered arrangement of chromosomes in the somatic nucleus of common wheat Triticum aestivum L.

Spatial relationships between chromosomes of the same genome, both homologous and non-homologous, were studied in root-tip cells of common wheat, Triticum aestivum (2n = 6x = 42). Mean distance between members of all the 21 homologous pairs (seven in each of the three genomes) and of 45 out of the 63 possible non-homologous combinations of two (21 in each genome) were determined. To minimize disruption of nuclear chromosomal arrangement, the cells were pretreated with cold temperature either in tap water or in a physiological medium (White solution) and distances between cytologically marked chromosomes were measured at metaphase. Comparison of distances for homologues with those for non-homologues indicated clearly that, within each genome, the homologous chromosomes were significantly closer to one another than were the non-homologues. Distances between homologues were similar in all three genomes, as were distances between non-homologues. The data are consistent with the hypothesis that the chromosomes of each genome of common wheat are arranged in the somatic nucleus in a highly specific ordered pattern. In this hypothetical arrangement, homologous chromosomes are closely associated, while the nonhomologues occupy definite positions with respect to one another. The universality of the phenomenon and its cellular mechanism and biological significance are discussed.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

The Mechanism of Somatic Association in Common Wheat, TRITICUM AESTIVUM L. IV. Further Evidence for Modification of Spindle Tubulin through the Somatic-Association Genes as Measured by Vinblastine Binding

Treatment with the antitubulin vinblastine was found to disrupt the spindle system in dividing root-tip cells of common wheat, Triticum aestivum L. Genotypes lacking the somatic association suppressor gene on 5B^L, or containing the somatic-association promoter on 5B^S, were found to be more sensitive to the treatment. In genetic lines carrying the somatic association suppressor, sensitivity to vinblastine was lower and there was a direct correlation between dosage of the suppressor gene (0, 2, and 4) and the decrease in spindle disruption on exposure to various concentrations of vinblastine. It is concluded that the somatic association genes affect binding ability of spindle tubulin to vinblastine. Since the same genes affect binding of colchicine to tubulin and since the two alkaloids attach to different sites it is assumed that the somatic association suppressor gene has a broad effect on the tubulin molecules which is not confined to a single site. The relevance of genetic control of antitubulin binding to somatic association is discussed.     

Frameshift suppressor mutations outside the anticodon in yeast proline tRNAs containing an intervening sequence.

Extragenic suppressors of +1 frameshift mutations in proline codons map in genes encoding two major proline tRNA isoacceptors. We have shown previously that one isoacceptor encoded by the SUF2 gene (chromosome 3) contains no intervening sequence. SUF2 suppressor mutations result from the base insertion of a G within a 3'-GGA-5' anticodon, allowing the tRNA to read a 4-base code word. In this communication we describe suppressor mutations in genes encoding a second proline tRNA isoacceptor (wild-type anticodon 3'-GGU-5') that result in a novel mechanism for translation of a 4-base genetic code word. The genes that encode this isoacceptor include SUF7 (chromosome 13), SUF8 (chromosome 8), trn1 (chromosome 1), and at least two additional unmapped genes, all of which contain an intervening sequence. We show that suppressor mutations in the SUF7 and SUF8 genes result in G-to-U base substitutions at position 39 that disrupted the normal G . C base pairing in the last base pair of the anticodon stem adjacent to the anticodon loop. These anticodon stem mutations might alter the size of the anticodon loop and permit the use of a 3'-GGGU-5' sequence within the loop to read 4-base proline codons. Uncertainty regarding the exact structure of the mature suppressor tRNAs results from the possibility that anticodon stem mutations might affect sites of intervening sequence removal. The possible role of the intervening sequence in the generation of mature suppressor tRNA is discussed. Besides an analysis of suppressor tRNA genes, we have extended previous observations of the apparent relationship between tRNA genes and repetitive delta sequences found as solo elements or in association with the transposable element TY1. Hybridization studies and a computer analysis of the DNA sequence surrounding the SUF7 gene revealed two incomplete, inverted delta sequences that form a stem and loop structure located 165 base pairs from the 5' end of the tRNA gene. In addition, sequences beginning 164 base pairs from the 5' end of the trn1 gene also exhibit partial homology to delta. These observations provide further evidence for a nonrandom association between tRNA genes and delta sequences.     

Diversity of restriction-modification gene homologues in Helicobacter pylori

The complete genome sequences of two Helicobacter pylori strains have recently become available. We have searched them for homologues of restriction-modification genes. One strain (26695) carried 52 such homologues, and the other (J99) carried 53. Their sequence alignments were arranged in the form of a phylogenetic tree and compared with the tree based on rRNA. The trees showed that the homologues are scattered among diverse groups of bacteria. They also revealed high polymorphism within the species--there are 42 pairs with high homology, 10 specific to 26695, and 11 specific to J99. Many of the restriction-modification homologues were characterized by a GC content lower than that of the average gene in the genome. Some of the restriction-modification homologues showed a different codon use bias from the average genes. These observations are interpreted in terms of horizontal transfer of the restriction-modification genes.     

The mechanism of meiotic homologue pairing

Homologous chromosome pairing involves the moving together of matching chromosomes or chromosome segments across substantial distances within a nucleus. Although the time in the life cycle of initial association of homologues varies among organisms, it may well be that similar underlying mechanisms for its occurrence prevail throughout sexually reproducing eukaryotes. The means by which pairing its accomplished is in no case understood. In the apparent absence of a long range specific force of attraction, simple partial models have been proposed which relay for the most part upon interactions of chromosome ends (telomeres) with specialized portions of the nuclear envelope. While such interactions, as well as the persistence of chromosome orientation established by mitotic anaphase poleward movement of centromere regions, may provide in many cases for closer than random positioning of some parts of homologues, the distances remaining to be traversed are still long range in physical-chemical terms. Also, the specific pairing observed in some kinds of rearranged segments is not facilitated by such circumstances, even if synapsis is initiated at available homologous telomere pairs and proceeds to completion by a "zip-up" process. A unified, more complex model is considered which is designed to accommodate the various relevant findings. It invokes the interaction of intranuclear structures with intercalary and/or terminal chromosomal pairing sites, e.g. filamentous structures which specifically bind to these, and a contractile system involving proteins such as actin and myosin to draw homologues together.     

Characterization of Hordeum vulgare - Hordeum bulbosum chromosome substitution lines by restriction fragment length polymorphism analysis.

Plants obtained from crosses between Hordeum vulgare and H. bulbosum were previously analyzed cytologically and for isozyme composition. They were identified as possessing substitutions of one or more H. vulgare chromosomes by their H. bulbosum homoeologues. To confirm their constitution and assess the merits of molecular techniques, chromosome-specific probes developed for the Triticeae were hybridized to Southern blots of DNA extracted from these plants and their parents. The hybridization patterns in the substitution plants confirmed that particular chromosomes of H. vulgare were replaced by their H. bulbosum homoeologues. For most probes, heterozygosity between pairs of H. bulbosum chromosomes was recorded. A possible duplication involving H. bulbosum homoeologues of barley chromosomes 4 and 7 was observed. Although molecular and cytological methods for analyzing chromosomally engineered plants are complementary, molecular probes may uncover differences not discernible using light microscopy or isozyme analysis.     

Leucine aminopeptidase during meiotic development.

We found a leucine aminopeptidase (LAP; EC 3.4.11.1) to be abundant in meiotic prophase tissue of a basidiomycete, Coprinus cinereus. After direct purification of the aminopeptidase component from meiocytes, we cloned the gene by degenerate PCR using partial amino-acid sequences of the purified enzyme and 5' and 3' RACE. It was homologous to the eukaryotic leucine aminopeptidase gene. The recombinant protein possesses the characteristic activities of a Coprinus leucine aminopeptidase (CoLAP) with a molecular mass of 52.4 kDa, and forms a homohexamer. Northern blot and spatial distribution analysis by immunohistochemical staining indicated CoLAP to be abundant in meiotic prophase cells and the supporting cells around meiocytes, but scarce in mycelium cells. Interestingly, from zygotene to pachytene, CoLAP was mostly present in supporting cells around meiocytes, but from diplotene onwards, it was plentiful in meiocytes themselves, suggesting that its expression is required to control some of the biochemical events at meiotic prophase. Moreover, the strong expression of CoLAP mRNA immediately after treatment with methyl methanesulfonate in mycelium implies that CoLAP has a role in somatic DNA repair.     

Use of monozygotic twins in search for breast cancer susceptibility loci.

We have used Swedish monozygotic twins concordant for breast cancer to study genetic changes associated with the development of breast cancer. Because loss of heterozygosity (LOH) at a specific genomic region may reflect the presence of a tumour suppressor gene, loss of the same allele in both of the twins concordant for breast cancer may pinpoint a tumour suppressor gene that confers a strong predisposition to breast cancer. DNA samples extracted from the matched tumour and normal tissues of nine twin pairs were analysed for allelic imbalance using a set of microsatellite markers on chromosomes 1, 13, 16 and 17, containing loci with known tumour suppressor genes. The two main regions, where more twin pairs than expected had lost the same allele, were located at 16qtel', including markers D16S393, D16S305 and D16S413, and at 17p13, distal to the p53 locus. Our results show that the monozygotic twin model can be used to suggest candidate regions of potential tumour suppressor genes, even with a limited number of twin pairs.     

Patterns of nucleotide substitution among simultaneously duplicated gene pairs in Arabidopsis thaliana

We characterized rates and patterns of synonymous and nonsynonymous substitution in 242 duplicated gene pairs on chromosomes 2 and 4 of Arabidopsis thaliana. Based on their collinear order along the two chromosomes, the gene pairs were likely duplicated contemporaneously, and therefore comparison of genetic distances among gene pairs provides insights into the distribution of nucleotide substitution rates among plant nuclear genes. Rates of synonymous substitution varied 13.8-fold among the duplicated gene pairs, but 90% of gene pairs differed by less than 2.6-fold. Average nonsynonymous rates were approximately fivefold lower than average synonymous rates; this rate difference is lower than that of previously studied nonplant lineages. The coefficient of variation of rates among genes was 0.65 for nonsynonymous rates and 0.44 for synonymous rates, indicating that synonymous and nonsynonymous rates vary among genes to roughly the same extent. The causes underlying rate variation were explored. Our analyses tentatively suggest an effect of physical location on synonymous substitution rates but no similar effect on nonsynonymous rates. Nonsynonymous substitution rates were negatively correlated with GC content at synonymous third codon positions, and synonymous substitution rates were negatively correlated with codon bias, as observed in other systems. Finally, the 242 gene pairs permitted investigation of the processes underlying divergence between paralogs. We found no evidence of positive selection, little evidence that paralogs evolve at different rates, and no evidence of differential codon usage or third position GC content.     

A MULTILOCUS TEST OF SIMULTANEOUS DIVERGENCE ACROSS THE ISTHMUS OF PANAMA USING SNAPPING SHRIMP IN THE GENUS ALPHEUS

The completion of the Panamanian Isthmus is one of the greatest natural experiments in evolution, sending multiple species pairs from a broad range of taxonomic groups on independent evolutionary trajectories. The resulting transisthmian sister species have been used as model systems for examining consequences that accompany cessation of gene flow in formerly panmictic populations. However, variance in pairwise genetic distances of these "geminates" often exceeds expectations, seemingly conflicting with the assumption that separation of populations was contemporaneous with the final closure of the Isthmus. Multilocus datasets and coalescent-based analytical methods can be used to estimate divergence times while accounting for variance in gene divergence that predates isolation, thus removing the need to invoke unequal divergence times. Here we present results from Bayesian analyses of sequence data from seven nuclear and one mitochondrial marker in eight transisthmian species pairs in the snapping shrimp genus Alpheus . Divergence times in two species pairs were shown to occur much earlier than the Isthmus final closure, but much of the variance in pairwise genetic distances from cytochrome oxidase I (COI) was explained when ancestral polymorphisms were accounted for. Results illustrate how coalescent approaches may be more appropriate for dating recent divergences than for estimating ancient speciation events.