Evidence that recombination between reiterated sequences in the Bacillus subtilis chromosome does not occur via unequal crossing over.

An experimental system was designed to permit the detection of recombination events occurring via unequal crossing over between sister bacterial chromosomes in Bacillus subtilis. It exploits the fact that during spore development, genetic and metabolic cooperation occurs between two different cell types, only one of which survives. During the early stages of sporulation, the two chromosomes of the developing sporangiole lie in the same cell and recombination between them is possible, in principle. Internal duplications flanking a selectable antibiotic-resistance gene have been introduced into the spoIIIC, spoIVA and spoVJ genes, whose correct expression in the mother cell (non-surviving compartment) is necessary for completion of spore development. After incubation in a sporulation-inducing medium in the absence of selective pressure, these strains sporulate at a low frequency and up to 30% of the progeny are Spo-. They result from mosaic sporangioles, in which only the chromosome segregated into the mother cell compartment of the developing sporangiole contains a reconstituted spo gene. In mosaic sporangioles generated by unequal crossing over between sister bacterial chromosomes, the insertionally inactivated spo gene, segregated into the pre-spore compartment, would carry an extra copy of the duplication initially present. Analysis of the products of 124 independent recombination events giving rise to mosaic sporangioles provided no evidence for the occurrence of unequal crossing over.     

Cutting edge: expansion of the KIR locus by unequal crossing over

The killer Ig-like receptor (KIR) genes have high sequence similarity and are organized in a head-to-tail fashion. These properties may enhance misalignment of homologous chromosomes during synapsis preceding meiotic recombination, resulting in unequal crossing over. We have identified an extended KIR haplotype that contains a novel hybrid gene and two copies of each of two previously described KIR genes. A parsimonious mechanism for the derivation of this haplotype invokes unequal crossing over between two known ancestral KIR haplotypes. These data raise the possibility that unequal crossing over may be responsible in part for the expansion/contraction of KIR haplotypes as well as other homologous gene families that map in tandem.     

Satellite DNA and heterochromatin variants: The case for unequal mitotic crossing over

Summary Variations of constitutive heterochromatin (heteromorphisms) appear to be a general feature of eucaryotes. A variety of molecular and cytogenetic evidence supports the hypothesis that heteromorphisms result from unequal double-strand exchanges during mitotic DNA replication. Constitutive heterochromatin consists of highly repeated DNA sequences that are not transcribed. Thus, heteromorphisms are tolerated without overt phenotypic effect. Several of the highly repeated DNAs that comprise constitutive heterochromatin have been shown to contain site-specific endonuclease recognition sequences interspersed at regular intervals dependent upon nucleosome structure. These interspersed short repeated sequences could mediate unequal crossovers, resulting in quantitative variability of constitutive heterochromatin and satellite DNA. De novo variations of constitutive heterochromatin may be useful as markers of exposure to mutagens and/or carcinogens.     

Genetic mechanism of cis-AB inheritance. I. A case associated with unequal chromosomal crossing over.

In contradiction to the general Mendelian inheritance of blood group ABO expression, the A and B characteristics are inherited together from one parent in the rare Cis-AB phenotype. Since the synthesis of blood group A and B substances are controlled by N-acetylgalactosoaminyltransferase (A-enzyme) and galactosyltransferase (B-enzyme), the genetic mechanism of Cis-AB expression may be elucidated by examining the characteristics of A- and B-enzymes in Cis-AB plasma. Biochemical study reveals that the examined Cis-AB plasma contains two separable enzyme components: one with kinetic properties similar to those of common A2-enzyme, but differing from A1-enzyme, and another with kinetic characteristics similar to those of common B-enzyme. Therefore, Cis-AB expression, at least in the case examined, is due to unequal crossing over, producing a chromosome with alleles for A2- and B-enzymes, rather than to a structural mutation in A or B alleles producing a single abnormal enzyme with bifunctional activity.     

Evidence for separate genetic control of crossing over and chiasma maintenance in maize

The meiotic cytological behavior of chromosomes in maize microsporocytes homozygous for the recessive mutant desynaptic was studied at various stages. It was found that following apparently normal pachytene synapsis there appears to be sporadic precocious desynapsis. By diakinesis bivalents heterozygous for a distal knob have often separated to pairs of univalents, each with a knob-carrying and a knobless chromatid. From the frequency of such events it is inferred that the crossover process is probably not affected by the mutant and that the genetic defect affects instead a distinct function concerned with chiasma maintenance following crossing over. Since precocious separation of dyads to monads at prophase II was also found in the desynaptic material, it is suggested that normal chiasma maintenance until anaphase I and normal dyad integrity maintenance between anaphase I and anaphase II may depend upon the same mechanism; it is also suggested that this may involve a special tendency for cohesiveness of sister chromatids during meiosis, beyond that which is ordinarily found at mitosis.     

Sequence homologies of diverse length tandem repetitions near ends of vaccinia virus genome suggest unequal crossing over.

The 180,000 base pair (bp), covalently closed, linear duplex DNA genome of vaccinia virus contains a 10,000 bp inverted terminal repetition within which are one set of 13 and one set of 18 tandem 70 bp repeating units. A 967 bp segment containing the innermost 70 bp repeat and an adjacent region notable for a scarcity of restriction endonuclease sites has been sequenced. This was facilitated by the cloning of TaqI and partial TaqI fragments in pBR322. We found that the innermost 70 bp repeat overlaps one of two adjacent 125 bp repeats, following which are eight repeats of 54 bp, parts of 54 bp and 70 bp repeats, and four consecutive 6 to 7 bp repeats. The 70, 125, and 54 bp repeating units have extensive sequence homologies and redundancies that suggest evolution by unequal crossing over. Schemes whereby unequal crossovers of 54 bp repeats lead to a recombinant segment 86% homologous to the 125 bp repeat and unequal crossovers of 125 bp repeats lead to a recombinant segment 94% homologous to the 70 bp repeat were considered. This propensity for sequence divergence should provide a useful marker for comparing the relatedness of poxviruses.     

An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

A monoclonal antibody specific for the 200 K polypeptide of the neurofilament triplet

A mouse monoclonal antibody, designated NF1, was obtained from a cloned hybridoma isolated from a fusion of mouse myeloma Sp2 cells with spleen cells from a BALB/c mouse immunized with a crude neurofilament preparation from porcine spinal cord. NF1 is an IgG1 and recognizes, in immune blotting procedures, only the 200 K neurofilament triplet component. Its neurofilament-specific nature is further revealed by immunofluorescence microscopy studies on frozen tissue sections and various cultured cells. Immunoelectron microscopy studies on cytoskeletons of cultured neurones emphasize the discontinuous display along each neurofilament previously observed with polyclonal antibodies specific for the 200 K component after appropriate but rather cumbersome cross-absorption steps. Use of NF1 on various neuronal cells strongly supports the previous proposal of the existence of certain subpopulations of neurofilament-free neurones and the observation that certain neuronal arrangements, (e.g., those in dendrites of pyramidal cells of the hippocampus), although rich in neurofilaments, probably lack the normal 200 K triplet component. Since NF1 shows a broad cross-species reactivity and is able to react on formaldehyde-fixed tissue, it should be a useful reagent to study differential neurofilament expression and organization in embryonic, adult and pathological tissues.     

Isolation of a cDNA for the rat heavy neurofilament polypeptide (NF-H)

We have isolated from a rat brain lambda gt11 expression library two overlapping cDNA clones of sizes 2.5 and 3.0 kb corresponding to the heavy neurofilament polypeptide (NF-H). The 2.5 kb insert apparently represents virtually the whole of the C-terminal tail, the 3.0 kb insert also encodes the conserved epitope for the monoclonal antibody, anti-IFA. The identity of the cDNAs was established by comparison of the predicted amino acid sequence with the known partial amino acid sequence of porcine NF-H. A repeat peptide sequence that may be a multiphosphorylation site was identified in the C-terminal non-helical tail.     

X-Y crossing over in the chimpanzee

Summary Single-copy DNA sequences defining several pseudoautosomal loci on the human sex chromosomes are shown to be highly conserved in the genome of the chimpanzee. Segregation analysis of polymorphic pseudoautosomal probes in a chimpanzee pedigree revealed that the transmission of the paternal alleles was not strictly sex-linked. In situ hybridization localized the pseudoautosomal probe 29C1 specifically to Xp22-Xpter and to Yq12.2-Yqter on the chimpanzee sex chromosomes. Thus, our results demonstrate the existence of homologous segments on the chimpanzee X and Y chromosomes, which regularly undergo recombinatory exchange in male meiosis. The chimpanzee is now the third mammalian species, besides man and mouse, in which there is genetic evidence for a pseudoautosomal segment on the sex chromosomes.     

Evidence that nucleotide sequence identity is a requirement for meiotic crossing over within the mouse Eb recombinational hot spot.

Meiotic recombination in the mouse is sometimes restricted to specific chromosomal sites. For example, when recombinants within the I region of the mouse major histocompatibility complex (MHC) are examined, the breakpoints between standard alleles can usually be mapped to the Eb gene. DNA sequence analysis of five cases of meiotic crossing over associated with this gene suggests that the recombinational hot spot may be confined to large regions of nucleotide identity located within the second intron of the Eb gene.     

Control of crossing over

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.     

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.     

Role of neurofilament light polypeptide in head and neck cancer chemoresistance

Resistance to cisplatin-based chemotherapy is responsible for therapeutic failure of many common human cancers including cancer of head and neck (HNC). Mechanisms underlying cisplatin resistance remain unclear. In this study, we identified neurofilament light polypeptide (NEFL) as a novel hypermethylated gene associated with resistance to cisplatin-based chemotherapy in HNC. Analysis of 14 HNC cell lines revealed that downregulation of NEFL expression significantly correlated with increased resistance to cisplatin. Hypermethylation of NEFL promoter CpG islands was observed in cell lines as examined by bisulfite DNA sequencing and methylation-specific PCR (MSP) and tightly correlated with reduced NEFL mRNA and protein expression. Furthermore, in patient samples with HNC (n = 51) analyzed by quantitative MSP, NEFL promoter hypermethylation was associated with resistance to cisplatin-based chemotherapy [relative risk (RR), 3.045; 95% confidence interval (CI), 1.459-6.355; P = 0.007] and predicted diminished overall and disease-free survival for patients treated with cisplatin-based chemotherapy. Knockdown of NEFL by siRNA in the highly cisplatin-sensitive cell line PCI13 increased (P < 0.01) resistance to cisplatin. In cisplatin-resistant O11 and SCC25cp cells, restored expression of NEFL significantly increased sensitivity to the drug. Furthermore, NEFL physically associated with tuberous sclerosis complex 1 (TSC1), a known inhibitor of the mTOR pathway, and NEFL downregulation led to functional activation of mTOR pathway and consequentially conferred cisplatin resistance. This is the first study to show a role for NEFL in HNC chemoresistance. Our findings suggest that NEFL methylation is a novel mechanism for HNC chemoresistance and may represent a candidate biomarker predictive of chemotherapeutic response and survival in patients with HNC.     

Evidence for a structurally homologous Rh-like polypeptide in Rhnull erythrocytes.

Human Rhnull red blood cells fail to react with Rh antibodies, indicating that these cells are either devoid of Rh protein or, like other species, possess antigenically distinct variants. To determine whether Rhnull cells possess an Rh-like polypeptide, 32-kDa proteins from D--, rr, and Rhnull cells were labeled with the cysteine-specific probe, 125I-labeled pyridyldithioethylamine. Size comparisons of labeled proteins in Triton X-100-solubilized membranes from Rh-bearing and Rhnull cells showed similar sedimentation coefficients and Stoke's radii. Immunoprecipitated Rh(D) from D-- cells, Rh(c) from rr cells, and purified 32-kDa proteins from Rhnull cells were digested with alpha-chymotrypsin and examined by high-performance liquid chromatography and by two-dimensional iodopeptide mapping. Analysis of 125I-labeled chymotryptic fragments from immunoprecipitated Rh(D) and Rh(c) showed the labeled peptides from both phenotypes to be virtually identical. High-performance liquid chromatography profiles and iodopeptide maps of 32-kDa Rhnull proteins yielded patterns identical to 32-kDa proteins isolated from D-- cells and rr cells with the exception of one missing 125I-labeled peptide. Further analysis of the Rh-related fragments from Rhnull cells showed significant homology with immunoprecipitated Rh(D) and Rh(c). DNA sequence analysis of cysteine-encoding regions from Rh-bearing and Rhnull cells showed complete identity. These data suggest that Rhnull red blood cells, although serologically distinct, possess an Rh-like protein that is structurally very similar to Rh(D) and Rh(c).     

Chromosome-wide control of meiotic crossing over in C. elegans

A central event in sexual reproduction is the reduction in chromosome number that occurs at the meiosis I division. Most eukaryotes rely on crossing over between homologs, and the resulting chiasmata, to direct meiosis I chromosome segregation, yet make very few crossovers per chromosome pair. This indicates that meiotic recombination must be tightly regulated to ensure that each chromosome pair enjoys the crossover necessary to ensure correct segregation. Here, we investigate control of meiotic crossing over in Caenorhabditis elegans, which averages only one crossover per chromosome pair per meiosis, by constructing genetic maps of end-to-end fusions of whole chromosomes. Fusion of chromosomes removes the requirement for a crossover in each component chromosome segment and thereby reveals a propensity to restrict the number of crossovers such that pairs of fusion chromosomes composed of two or even three whole chromosomes enjoy but a single crossover in the majority of meioses. This regulation can operate over physical distances encompassing half the genome. The meiotic behavior of heterozygous fusion chromosomes further suggests that continuous meiotic chromosome axes, or structures that depend on properly assembled axes, may be important for crossover regulation.     

Age-related changes in crossing over in Drosophila resemble the picture of interchromosomal effect of chromosome rearrangement on crossing over

Crossing over in the left arm of chromosome 2 (2L) was studied in successive broods of Drosophila melanogaster females carrying intact chromosomes (+/+), inversion Muller-5 in the X chromosome (M-5/+), and insertion of the Y-chromosome material into region 34A (Is(2L)/+). The regions net-dp, dp-b, b-pr and pr-cn were examined in 14 two-day-old broods of females +/+ and M-5/+ and in 10 broods of females Is(2L)/+. In all lines, the highest level of crossing over was in the first three broods (eggs laid during the first 6 days of oviposition) and the lowest level in the broods 7-8 (eggs laid at days 14-16). A high rate of crossing over in the first broods of females +/+ and M-5/+ was due to an increment of exchanges in the proximal euchromatin regions (b-pr and pr-cn) and to an increase in the number of tetrads with double exchanges. These changes are similar to a pattern of the interchromosomal effect on crossing over (IEC) in structurally normal chromosomes. In Is(2L)/+ females, a high level of crossing over was due to extensive exchanges in the interstitial regions net-dp and dp and an increase in the number of tetrads with single exchanges. These changes resembled the IEC in rearranged chromosomes (in this case, in chromosomes bearing an insertion). Thus, the age changes of crossing over are similar to the consequences of the presence or absence of IEC. Age changes in crossing over in a chromosome depended both on the local rearrangements in this chromosome (the local effect on crossing over, LEC) and on rearrangements in nonhomologous chromosomes (IEC). In the first broods, both LEC and IEC decreased with an increase in the level of crossing over. In subsequent broods, the reduced level of crossing over was accompanied by an increase in both LEC and IEC. This suggests that the mechanisms responsible for the age changes in crossing over and IEC may have common steps. The contact model of crossing over may explain the similarity between the age changes in crossing-over and IEC. It is suggested that both phenomena result from delayed determination of crossing over in a meiotic cell. This may occur due to the retarded formation of the local contacts in one of the homologous chromosome pairs or because a higher number of local contacts is required to trigger crossing over in a meiotic cell (of early age).