Efficient nonhomologous and homologous recombination in scid cells

The severe combined immunodeficiency (scid) mutation affects both coding joint formation during immunoglobulin and T-cell receptor V(D)J recombination and double-strand break repair. We analyzed scid cells for their ability to undergo other types of DNA end joining: non-homologous and homologous recombination. Using plasmid constructs carrying antibiotic resistance genes, we observed that the efficiency of nonhomologous integration in scid cells was equal to that in wildtype cell lines. In addition, there was no obvious difference in the fidelity of the integration and in the expression of the resistance genes. Moreover, scid cells were able to carry out homologous recombination of extrachromosomal substrates just as well as wildtype cells. These results suggest a mechanistic difference between nonhomologous integration and homologous recombination on the one hand and V(D)J recombination and double-strand break repair on the other.     

Homologous and non-homologous chromosome associations by interchromosomal chromatin connectives in Ornithogalum virens

Based on interchromosomal chromatin connectives, a statistical analysis of homologous and non-homologous chromosome associations was made on mitotic metaphase chromosomes of Ornithogalum virens. The great majority of connectives involve constitutive heterochromatin, and connections between homologous chromosomes are twice as common as would be expected by chance. It is suggested that constitutive heterochromatin with similar DNA is involved in both homologous and non-homologous chromosome associations.     

Non-recurrent 17p11.2 deletions are generated by homologous and non-homologous mechanisms

Several recurrent common chromosomal deletion and duplication breakpoints have been localized to large, highly homologous, low-copy repeats (LCRs). The mechanism responsible for these rearrangements, viz., non-allelic homologous recombination between LCR copies, has been well established. However, fewer studies have examined the mechanisms responsible for non-recurrent rearrangements with non-homologous breakpoint regions. Here, we have analyzed four uncommon deletions of 17p11.2, involving the Smith–Magenis syndrome region. Using somatic cell hybrid lines created from patient lymphoblasts, we have utilized a strategy based on the polymerase chain reaction to refine the deletion breakpoints and to obtain sequence data at the deletion junction. Our analyses have revealed that two of the four deletions are a product of Alu/Alu recombination, whereas the remaining two deletions result from a non-homologous end-joining mechanism. Of the breakpoints studied, three of eight are located in LCRs, and five of eight are within repetitive elements, including Alu and MER5B sequences. These findings suggest that higher-order genomic architecture, such as LCRs, and smaller repetitive sequences, such as Alu elements, can mediate chromosomal deletions via homologous and non-homologous mechanisms. These data further implicate homologous recombination as the predominant mechanism of deletion formation in this genomic interval.     

A musculoskeletal model of the hand and wrist: model definition and evaluation

Abstract

To improve our understanding on the neuromechanics of finger movements, a comprehensive musculoskeletal model is needed. The aim of this study was to build a musculoskeletal model of the hand and wrist, based on one consistent data set of the relevant anatomical parameters. We built and tested a model including the hand and wrist segments, as well as the muscles of the forearm and hand in OpenSim. In total, the model comprises 19 segments (with the carpal bones modeled as one segment) with 23 degrees of freedom and 43 muscles. All required anatomical input data, including bone masses and inertias, joint axis positions and orientations as well as muscle morphological parameters (i.e. PCSA, mass, optimal fiber length and tendon length) were obtained from one cadaver of which the data set was recently published. Model validity was investigated by first comparing computed muscle moment arms at the index finger metacarpophalangeal (MCP) joint and wrist joint to published reference values. Secondly, the muscle forces during pinching were computed using static optimization and compared to previously measured intraoperative reference values. Computed and measured moment arms of muscles at both index MCP and wrist showed high correlation coefficients (r?=?0.88 averaged across all muscles) and modest root mean square deviation (RMSD?=?23% averaged across all muscles). Computed extrinsic flexor forces of the index finger during index pinch task were within one standard deviation of previously measured in-vivo tendon forces. These results provide an indication of model validity for use in estimating muscle forces during static tasks.     

Supernumerary segments in five species of grasshoppers (Orthoptera: Acridoidea)

Supernumerary segments have been observed in five species of grasshoppers: Calliptamus barbarus, Oedipoda fuscocincta, Acrotylus insubricus, Omocestus raymondi and Chorthippus ariasi. In four cases they are located in the S-chromosomes, but in A. insubricus they are carried by the megameric pair (M₉). In C. barbarus and O. fuscocincta we have observed non-homologous association during diplotene between the extra segments and the X-chromosome. The supernumerary segments of these two species are distally located. However, in anaphase-I the unequal bivalents divide reductionally in 30% and 20% of the cells, respectively. Finally, the supernumerary segments of C. barbarus do not produce any effect on mean chiasma frequency, but they decrease significantly the between-cell variance of chiasmata in males carrying them.     

Redistribution of intra- and inter-limb support moments during downhill walking on different slopes

Downhill walking presents a greater risk of falling as a result of slipping or loss of balance in comparison with level walking. The current study aimed to investigate the effects of inclination angles on the intra-limb (inter-joint) and inter-limb sharing of the body support during downhill walking for a better understanding of the associated control strategy. Fifteen young male adults (age: 32.6±5.2 years, height: 168.9±5.5 cm, mass: 68.4±8.7 kg) performed level and downhill walking while their kinematic and kinetic data were measured for calculating joint moments and total support moments of the lower limbs using inverse dynamics analysis. The peak total support moments of both the leading and trailing limbs increased with increasing inclination angles (p<0.05) with different sharing patterns among individual joints. Being the major contributor to the peak total support moment during early single-limb support, the contribution of the knee remained unaltered (p>0.05), but the contributions of the hip increased with reduced contributions from the ankle (p<0.05). For the increased peak total support moment during late single-limb support, the intra-limb sharing changed from a major ankle contribution to a major knee contribution strategy. The hip contribution was also increased (p<0.05) but the hip flexor moment remained unaltered (p>0.05). During double-limb support, the main contributor to the whole body support changed from the trailing limb to the leading limb with increasing inclination angles (p<0.05).     

Bilateral organization of physiological tremor in the upper limb

The bilateral patterns of physiological tremor in the upper limb of adults were examined under conditions where eight combinations of the elbow, wrist and index-finger joints of the right arm were braced using individually molded splints. The hypotheses tested were that: (a) coordination of upper-limb tremor involves (compensatory) coupling of intra- but not inter-limb segments, (b) splinting the respective joints of the right arm changes the organization of this synergy in both limbs, and (c) reducing the involvement of joint-space degrees of freedom through restricting their motion (by splinting) results in increased tremor in the distal segments. Under no-splinting conditions, significant relationships were only observed between adjacent (intra-limb) effector units, with the strength of the correlation increasing from proximal to distal. Splinting the right limb resulted in an increase in the strength and number of significant intra-limb relationships in both limbs. No inter-limb tremor relationships were found between any segment during this task, irrespective of the splinting condition. The frequency profile for the tremor in each limb segment showed two prominent frequency peaks (at 2-4 Hz and 8-12 Hz). A third, higher frequency peak (18-22 Hz) was observed in the index fingers only. Splinting the right limb produced a general increase in the amplitude and variability of tremor in the fingertip of both arms. This effect was particularly strong under conditions where the more proximal joints were splinted. The lack of any between-limb relationships, coupled with the fact that splinting one limb influenced both limbs, suggests that some form of linkage does exist between the limbs. It is unlikely that mechanical linkages can explain fully these relationships. It is proposed that the tremor observed in either limb represents the output of a central oscillatory mechanism(s), but that this output is subsequently independently filtered in a parallel fashion on its way to each respective limb. A common bilateral (compensatory) strategy is employed to minimize the tremor in either limb during this multiple-degrees-of-freedom task.     

Effects of bilateral medial knee osteoarthritis on intra- and inter-limb contributions to body support during gait

Patients with knee OA show altered gait patterns, affecting their quality of living. The current study aimed to quantify the effects of bilateral knee OA on the intra-limb and inter-limb sharing of the support of the body during gait. Fifteen patients with mild, 15 with severe bilateral knee OA, and 15 healthy controls walked along a walkway while the kinematic and kinetic data were measured. Compared with the controls, the patients significantly reduced their knee extensor moments and the corresponding contributions to the total support moment in the sagittal plane (p<0.05). For compensation, the mild OA group significantly increased the hip extensor moments (p<0.05) to maintain close-to-normal support and a more symmetrical inter-limb load-sharing during double-limb support. The severe OA group involved compensatory actions of both the ankle and hip, but did not succeed in maintaining a normal sagittal total support moment during late stance, nor a symmetrical inter-limb load-sharing during double-limb support. In the frontal plane, the knee abductor moments and the corresponding contributions to the total support moment were not affected by the changes in the other joints, regardless of the severity of the disease. The observed compensatory changes suggest that strengthening of weak hip muscles is essential for body support during gait in patients with knee OA, but that training of weak ankle muscles may also be needed for patients with severe knee OA.     

The size and ratio of homologous sequence to non-homologous sequence in gene disruption cassette influences the gene targeting efficiency in <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Targeted gene replacement via homologous recombination (HR) is a conventional approach for the analysis of gene function. However, this event is rare in Beauveria bassiana, which hampers efficient functional analysis in this widely used entomopathogenic fungus. To improve homologous recombination frequency in B. bassiana, we investigated the effect of the ratio of homologous sequence to non-homologous sequence (HS/NHS) in gene disruption cassette upon the HR frequency by two gene loci BbNtl and BbThi, using the herpes simplex virus thymidine kinase as a negative selectable marker against ectopic transformants. Our data revealed that an increase of the ratio of HS/NHS achieved by either extending homologous sequence or decreasing non-homologous sequence could improve HR frequency in B. bassiana. We determined empirically that (1) at least 700 bp of homology to both sides of a target gene was needed to get a reasonable number of disruptants, e.g., 6.7‰ to 13.3‰ in B. bassiana. (2) When the ratio of HS/NHS was above 0.8, an acceptable HR frequency could be achieved for gene replacement in B. bassiana, while when the ratio was below 0.3, few gene disrupted mutants were obtained.     

RAD51 supports spontaneous non-homologous recombination in mammalian cells, but not the corresponding process induced by topoisomerase inhibitors

The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining.     

Meiotic chromosome synapsis in a haploid yeast

An extensive synaptonemal complex (SC) is found at pachytene in whole mount spread preparations of a haploid yeast, Saccharomyces cerevisiae, strain. Whereas unsynapsed axial elements are present only in a few nuclei, in others non-homologous synapsis involves virtually the whole chromosome set. This suggests that homology is not an indispensable precondition for SC formation in yeast but that chromosomes engage in non-homologous synapsis if no homologous partner is available. Recent evidence that in the sporulation deficient yeast mutants rad50 and mer1 axial elements do form but remain unsynapsed in the majority of nuclei is discussed in the light of the above findings.by D. Schweizer     

Synaptic adjustment,non-homologous pairing,and non-pairing of homologous segments in sex chromosome mutants of Ephestia kuehniella (Insecta,Lepidoptera)

Microspread pachytene nuclei of wild-type and W chromosome mutants of the mealmoth Ephestia kuehniella were used to study synaptonemal complex (SC) formation. In structurally heterozygous bivalents, axial elements of considerable length differences were brought to the same length by synaptic adjustment. The adjustment length was a compromise between the mutant and the wildtype homologue length in a structural heterozygote of a W chromosome-autosome translocation, T(A; W). The translocated non-homologous W segment really participated in SC formation as could be seen from the W chromosomal heterochromatin, used as a cytogenetic marker. Pachytene pairing of the wild-type W-Z bivalent extended from about two-thirds to the full length of the W chromosome, though from cytogenetic and genetic evidence W and Z are largely — if not completely — non-homologous. Nonhomologous pairing was even more conspicuous in sex chromosome bivalents containing a deleted W chromosome, Df(W). In one of the pairing configurations the halves of the Z chromosome were synapsed to either side of the Df(W). Thus, one side was pairing with the Df(W) in reversed order. The pairing behavior of the W with homologous chromosome segments was tested by introducing supernumerary W segments via the T(A; W) translocation. Pairing between the W and the translocated homologous W segment never occurred, whereas the Z frequently synapsed with it. Even in T(A; W) homozygotes, pairing between the two translocated W segments was not regularly found while the autosomal parts of the translocation chromosomes were always completely paired. Homologous chromosomes and the ability to form an SC are not sufficient for pairing initiation. Specific loci or sequences are postulated for this function. They are either absent from the W chromosome or are present in only low concentrations.     

Recognition sites and coordinate control of recombination in Schizophyllum

The genetic control of recombination in two chromosomal regions was studied in dikaryons of Schizophyllum commune with joint control of recombination in the A and B factors. The role of the controlled regions in the determination of their recombination frequencies was studied by substituting one or the other or both by homologous segments. The B factor was shown to affect both its own and the A factors' recombination frequency. A model of genetic control of recombination in several regions is proposed. The model is based on identical recognition sites within the jointly controlled segments. It is consistent with the fact that jointly-controlled segments can show either negative or positive correlation between their recombination frequencies.     

DNA Quadruplexes Assembled by Simple Peptide: Effects of DNA Homology and Peptide Removal

Abstract

Formation of heterologous (calf thymus dsDNA) and homologous (linearized pBR322 plasmid dsDNA) quadruplexes upon binding with the simple aliphatic tripeptide derivative (L- Val)₃?N₂H₂?DNS CF₃COOH—;DHTV) was examined by fluorimetry, flow linear (LD), circular dichroism (CD), and electron microscopy (EM). The morphology of the rod-like compact particles formed due to the association of dsDNA segments proved to be the same for both DNAs, whereas the stability of the compact DNA structure upon tripeptide removal from the complex with DNA differed substantially for homologous versus non-homologous dsDNA used. The increase in NaCl concentration in the solution up to 30 mM removes the peptide from both types of the complexes completely. At the same time at 20 mM NaCl calf thymus DNA quadruplexes readily dissociate, whereas the structures formed by plasmid DNA retain their morphology in the solution containing NaCl with concentrations up to 40 mM and are only partially disrupted at even higher NaCl concentration. These results provide an analogy between trivaline-DNA model complexes and RecA-DNA binding.     

Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger

In this study, the ku70 and ku80 homologs from the Aspergillus niger genome were identified and their function was analyzed using targeted mutagenesis. The role of the ku80 gene in non-homologous end-joining (NHEJ) was investigated by calculating the frequency of homologous recombination. The transformation test verified that the frequency of homologous recombination significantly increased, from 1.78 to 65.6% in ku80 single deletion strains and to 100% in ku70/ku80 double deletion strains. These results suggest that the ku80 gene is important for non-homologous end-joining. Although the morphology of the ku deletion strains colonies was similar to that of the wildtype strain, mutants were more sensitive to the mutagen phleomycin. Furthermore, the purified ku80 deletion strain produced some sectored colonies on hygromycin B-containing plates. This result suggests that the ku80 gene deletion leads to genomic instability in A. niger.     

Somatic association of chromosomes in asynaptic genotypes of Avena sativa L.

The distribution of distances between homologous chromosomes in root tip cells of Avena sativa was studied in synaptic and asynaptic genotypes. The distances between homologous chromosomes were smaller than that calculated for two randomly distributed chromosomes, while non-homologous chromosomes did not deviate from the random theoretical distribution. The distances between homologous chromosomes in the asynaptic genotype were significantly greater than in synaptic plants. The loose association of homologous chromosomes in somatic tissue is correlated with the failure of chromosome pairing at meiosis in asynaptic plants.     

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.     

INTERSPECIFIC HYBRIDIZATION IN HAPLOPAPPUS AND ITS BEARING ON CHROMOSOME EVOLUTION IN THE BLEPHARODON SECTION

Jackson , R. C. (U. Kansas, Lawrence.) Interspecific hybridization in Haplopappus and its bearing on chromosome evolution in the Blepharodon section. Amer. Jour. Bot. 49 (2) : 119–132. Illus. 1962.—Cytological analyses of interspecific hybrids between H. gracilis (n = 2) and H. spinulosus ssp. australis (n = 8) indicate that ssp. australis is a segmental allotetraploid, derived from past hybridization between 2 taxa with chromosome numbers of n = 4. Analysis of hybrids between H. gracilis (n = 2) and H. ravenii (n = 4), a previously undescribed species, has shown that the chromosome segments of these 2 species are almost completely homologous. Differential contraction is suggested as the explanation for the disappearance in late pachytene of presumed non-homologous segments which were evident in some cells at early pachytene. The pairing relationship of gracilis and ravenii chromosomes at pachytene and later prophase I stages of meiosis indicates that gracilis has evolved from ravenii by an aneuploid reduction process similar to that described for Crepis. The close morphological relationship of the 2 species adds further support to this proposition. Data from the cytological analysis of both interspecific hybrids indicate that x = 4 is the basic chromosome number for the Blepharodon section of Haplopappus.     

Somatic association of chromosomes in diploid and hexaploid Avena

The frequency distributions of certain homologous and non-homologous chromosomes in somatic cells of diploid and liexaploid species of Avena were studied and compared with the theoretical random distribution.All homologous chromosomes studied irrespective of shape and size showed non-random distribution in both diploid and hexaploid species. In all cases the homologous chromosomes were closer than would be expected with random distribution. Chromosomal characters such as size, shape, and presence or absence of nucleolar organizing regions did not exert appreciable influence on the somatic association of homologues.While the non-homologues followed the theoretical random distribution in diploid species, a significant deviation from the random curve was noted for non-homologues in hexaploid species. However, diploid and hexaploid non-homologous chromosomes had characteristic S-shaped cumulative frequency distributions which were distinct from the half-moon-shaped ones obtained for homologous chromosomes.The different regions (short arm, long arm, centromere and mid-points) of two pairs of homologous chromosomes (one of them being nucleolar) studied showed non-random distribution with the exception of the long arm of the non-nucleolar chromosome. From these results the role of the centromere or mutual attraction of homologous segments could not be assessed with certainty.Contribution No. 232 from the Research Station, Central Experimental Farm, Ottawa, Ontario, Canada.