PRINS-labeled knobs are not associated with increased chromosomal stickiness in the maize st1 mutant

In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.     

Human imprinted chromosomal regions are historical hot-spots of recombination

Human recombination rates vary along the chromosomes as well as between the two sexes. There is growing evidence that epigenetic factors may have an important influence on recombination rates, as well as on crossover position. Using both public database analysis and wet-bench approaches, we revisited the relationship between increased rates of meiotic recombination and genome imprinting. We constructed metric linkage disequilibrium (LD) maps for all human chromosomal regions known to contain one or more imprinted genes. We show that imprinted regions contain significantly more LD units (LDU) and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical "hot-spots" of recombination. We also demonstrate, by direct segregation analysis at the 11p15.5 imprinted region, that there is remarkable agreement between sites of meiotic recombination and steps in LD maps. Although the increase in LDU/Megabase at imprinted regions is not associated with any significant enrichment for any particular sequence class, major sequence determinants of recombination rates seem to differ between imprinted and control regions. Interestingly, fine-mapping of recombination events within the most male meiosis-specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions.     

Mutations in Escherichia coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids.

Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX+ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20 degrees C (J.R. Walker, J.A. Ramsey, and W.G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA+ allele restored growth to dnaA (Cs, Sx) strains at 20 degrees C independently of which allele was present on the plasmid. The dnaA (Cs, Sx) alleles provided initiator function at 39 degrees C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA (Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.     

Processed pseudogenes are located preferentially in regions of low recombination rates in the human genome

The aim of this article is to demonstrate possible recombination‐associated evolutionary forces affecting the genomic distribution of processed pseudogenes. The relationship between recombination rate and the distribution of processed pseudogenes is analysed in the human genome. The results show that processed pseudogenes preferentially accumulate in regions of low recombination rates and this correlation cannot be explained by indirect relationships with GC content and gene density. Several explanatory models for the observation are discussed. A model of selection against ectopic recombination is tested based on the difference in distribution pattern between two classes of processed pseudogenes, which differ in the possibility of stimulating ectopic recombination. Our results indicate that the correlation between processed pseudogene density and recombination rate is probably results, in part, from the selection against ectopic recombination between closely located homologous processed pseudogenes. We also found a length effect in processed pseudogene distribution, namely long processed pseudogenes are located more preferentially in regions of low recombination rates than short ones.     

The interaction of knobs and B chromosomes of maize in determining the level of recombination

Enhancement of recombination by B chromosomes is influenced by the kind of heterochromatic knob present in or near the tested region of the A chromosomes. In homomorphic chromosome 9 bivalents of K^s/K^s constitution, double exchanges were increased at the expense of singles, but in the K*/K^s heteromorphs there was a gain in both single and double exchanges at the expense of no-exchange tetrads. Modification of the B chromosome enhancement in different knob compounds was observed only in the megasporocytes.—Different frequencies of recombination are found in plants with odd and even numbers of B chromosomes; this effect is especially striking in the megasporocytes. The modification in recombination produced by an odd or even number of B chromosomes is a function of the interaction of a particular region and the knob constitution. Odd numbers of B chromosomes were more effective than even numbers in causing increased recombination.—It is concluded that heterochromatic knobs and the essentially heterochromatic supernumeraries may interact in the process of crossing over, with the level of recombination determined in part by knob constitution.     

Transposons but not retrotransposons are located preferentially in regions of high recombination rate in Caenorhabditis elegans

We analyzed the distribution of transposable elements (TEs: transposons, LTR retrotransposons, and non-LTR retrotransposons) in the chromosomes of the nematode Caenorhabditis elegans. The density of transposons (DNA-based elements) along the chromosomes was found to be positively correlated with recombination rate, but this relationship was not observed for LTR or non-LTR retrotransposons (RNA-based elements). Gene (coding region) density is higher in regions of low recombination rate. However, the lower TE density in these regions is not due to the counterselection of TE insertions within exons since the same positive correlation between TE density and recombination rate was found in noncoding regions (both in introns and intergenic DNA). These data are not compatible with a global model of selection acting against TE insertions, for which an accumulation of elements in regions of reduced recombination is expected. We also found no evidence for a stronger selection against TE insertions on the X chromosome compared to the autosomes. The difference in distribution of the DNA and RNA-based elements along the chromosomes in relation to recombination rate can be explained by differences in the transposition processes.     

Genes located in a chromosomal inversion are correlated with territorial song in white‐throated sparrows

The genome of the white‐throated sparrow (Zonotrichia albicollis) contains an inversion polymorphism on chromosome 2 that is linked to predictable variation in a suite of phenotypic traits including plumage color, aggression and parental behavior. Differences in gene expression between the two color morphs, which represent the two common inversion genotypes (ZAL2/ZAL2 and ZAL2/ZAL2^m), may therefore advance our understanding of the molecular underpinnings of these phenotypes. To identify genes that are differentially expressed between the two morphs and correlated with behavior, we quantified gene expression and terrirorial aggression, including song, in a population of free‐living white‐throated sparrows. We analyzed gene expression in two brain regions, the medial amygdala (MeA) and hypothalamus. Both regions are part of a ‘social behavior network’, which is rich in steroid hormone receptors and previously linked with territorial behavior. Using weighted gene co‐expression network analyses, we identified modules of genes that were correlated with both morph and singing behavior. The majority of these genes were located within the inversion, showing the profound effect of the inversion on the expression of genes captured by the rearrangement. These modules were enriched with genes related to retinoic acid signaling and basic cellular functioning. In the MeA, the most prominent pathways were those related to steroid hormone receptor activity. Within these pathways, the only gene encoding such a receptor was ESR1 (estrogen receptor 1), a gene previously shown to predict song rate in this species. The set of candidate genes we identified may mediate the effects of a chromosomal inversion on territorial behavior.     

Sites of recombination are local determinants of meiotic homolog pairing in Saccharomyces cerevisiae

Trans-acting factors involved in the early meiotic recombination pathway play a major role in promoting homolog pairing during meiosis in many plants, fungi, and mammals. Here we address whether or not allelic sites have higher levels of interaction when in cis to meiotic recombination events in the budding yeast Saccharomyces cerevisiae. We used Cre/loxP site-specific recombination to genetically measure the magnitude of physical interaction between loxP sites located at allelic positions on homologous chromosomes during meiosis. We observed nonrandom coincidence of Cre-mediated loxP recombination events and meiotic recombination events when the two occurred at linked positions. Further experiments showed that a subset of recombination events destined to become crossover products increased the frequency of nearby Cre-mediated loxP recombination. Our results support a simple physical model of homolog pairing in budding yeast, where recombination at numerous genomic positions generally serves to loosely coalign homologous chromosomes, while crossover-bound recombination intermediates locally stabilize interactions between allelic sites.     

Synapsis of recombination signal sequences located in cis and DNA underwinding in V(D)J recombination

V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.     

Restriction fragments homologous to mitochondrial plasmid-like DNAs are located within limited chromosomal regions on the rice nuclear genome

Summary The shifted multiplicative model (SHMM) is used with a cluster method to identify subsets of sites in an international maize (Zea mays L.) trial without genotypic rank-change. For cluster analysis, distance between two sites is defined as the residual sum of squares after fitting SHMM with one multiplicative term (SHMM₁) if SHMM₁ does not show genotypic rank-change. However, if SHMM₁ does show genotypic rank-change, the distance between two sites is defined as the smaller of the sums of squares owing to genotypes within each of the two sites. Calculation of distance between two sites is facilitated by using the site regression model with one multiplicative term (SREG₁), which can be reparameterized as SHMM₁ when only two sites are considered. The dichotomous splitting procedure, used on the dendrogram obtained from cluster analysis, will first perform SHMM analyses on each of the last two cluster groups to join (end of the dendrogram). If SHMM₁ does not give an adequate fit, the next step is to move down the branches of the tree until groups of sites (clusters) are found to which SHMM₁ provides an adequate fit and primary effects of sites are all of the same sign. Five final groups of sites to which SHMM₁ provides an adequate fit and primary effects of sites are all of the same sign were obtained. The procedure appears to be useful in identifying subsets of sites in which genotypic rank-change interactions are negligible.Research reported in this paper (Journal article no. 91-3-218) is part of a project of the Kentucky Agricultural Experiment Station, published with the approval of the Director     

Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

Accessibility of chromosomal recombination breaks in nuclei of wild-type and DNA-PKcs-deficient cells

V(D)J recombination is a highly regulated process, proceeding from a site-specific cleavage to an imprecise end joining. After the DNA excision catalyzed by the recombinase encoded by recombination activating genes 1 and 2 (RAG1/2), newly generated recombination ends are believed held by a post-cleavage complex (PC) consisting of RAG1/2 proteins, and are subsequently resolved by non-homologous end joining (NHEJ) machinery. The relay of these ends from PC to NHEJ remains elusive. It has been speculated that NHEJ factors modify the RAG1/2-PC to gain access to the ends or act on free ends after the disassembly of the PC. Thus, recombination ends may either be retained in a complex throughout the recombination process or left as unprotected free ends after cleavage, a condition that may permit an alternative, non-classical NHEJ end joining pathway. To directly test these scenarios on recombination induced chromosomal breaks, we have developed a recombination end protection assay to monitor the accessibility of recombination ends to exonuclease-V in intact nuclei. We demonstrate that these ends are well protected in the nuclei of wild-type cells, suggesting a seamless cleavage-joining reaction. However, divergent end protection of coding versus signal ends was found in cells derived from severe combined immunodeficient (scid) mice that are defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While signal ends are resistant, opened coding ends are susceptible to enzymatic modification. Our data suggests a role of DNA-PKcs in protecting chromosomal coding ends. Furthermore, using recombination inducible scid cell lines, we demonstrate that conditional protection of coding ends is inversely correlated with the level of their resolution, i.e., the greater the accessibility of the coding ends, the higher level of coding joints formed. Taken together, our findings provide important insights into the resolution of recombination ends by error-prone alternative NHEJ pathways.