Normal Homologous Chromosome Research Articles

FIGNL1 Inhibits Non-homologous Chromosome Association and Crossover Formation.

Meiotic crossovers (COs) not only generate genetic diversity but also ensure the accuracy of homologous chromosome segregation. Here, we identified FIGNL1 as a new inhibitor for extra crossover formation in rice. The fignl1 mutant displays abnormal interactions between non-homologous chromosomes at diakinesis, and chromosome bridges and fragmentation at subsequent stages of meiosis, but shows normal homologous chromosome pairing and synapsis during early prophase I. FIGNL1 participates in homologous chromosome recombination and functions downstream of DMC1. Mutation of FIGNL1 increases the number of bivalents in zip4 mutants, but does not change the number of HEI10 foci, indicating that FIGNL1 functions in limiting class II CO formation. FIGNL1 interacts with MEICA1, and colocalizes with MEICA1 in a dynamic pattern as punctate foci located between two linear homologous chromosomes. The localization of FIGNL1 depends on ZEP1-mediated assembly of the synaptonemal complex. Based on these results, we propose that FIGNL1 inhibits non-homologous chromosome interaction and CO formation during rice meiosis.

Male infertility associated with de novo pericentric inversion of chromosome 1.

Inversion occurs after two breaks in a chromosome have happened and the segment rotates 180° before reinserting. Inversion carriers have produced abnormal gametes if there is an odd number crossing- over between the inverted and the normal homologous chromosomes causing a duplication or deletion. Reproductive risks such as infertility, abortion, stillbirth and birth of malformed child would be expected in that case. A 54-year- old male patient was consulted to our clinic for primary infertility. The routine chromosome study were applied using peripheral blood lymphocyte cultures and analyzed by giemsa-trypsin-giemsa (GTG) banding, and centromer banding (C-banding) stains. Y chromosome microdeletions in the azoospermia factor (AZF) regions were analyzed with polymerase chain reaction. Additional test such as fluorescence in situ hybridization (FISH) was used to detect the sex-determining region of the Y chromosome (SRY). Semen analysis showed azoospermia. A large pericentric inversion of chromosome 1 46,XY, inv(1) (p22q32) was found in routine chromosome analysis. No microdeletions were seen in AZF regions. In our patient the presence of SRY region was observed by using FISH technique with SRY-specific probe. Men who have pericentric inversion of chromosome 1, appear to be at risk for infertility brought about by spermatogenic breakdown. The etiopathogenic relationship between azoospermia and pericentric inversion of chromosome 1 is discussed.

Trisomía parcial 9p derivada de una translocación recíproca 9;15, materna. Reporte de casos

Objectives: to highlight the importance of performing karyotype in children with congenital malformations in order to have a confirmatory diagnosis, in parents to exclude the possibility of being carriers of chromosomal abnormalities and perform the genetic counseling. Clinical cases description: Female patient with 3 years and 2 months old to whom karyotype was performed by global neurodevelopmental delay and microcephaly, and her mother with 34 years old without any clinical manifestations, to both patients, lymphocyte culture and chromosomal analysis with a High Resolution Banding techniques GTG and C were performed. The mother’s karyotype was 46,XX,t(9;15)(q10;q10)(p10;p10),add14p. The father’s karyotype was normal, 46,XY, and the girl’s karyotype resulted in a pure Trisomy 9p: 47,XX,+del(9)(q11). Discussion: This chromosomal rearrangement in mother included a nonhomologous reciprocal translocation between the long arms of pair chromosomes 9 and 15 and between the short arms of the same chromosomes, additional to it, an unknown origin material was also observed in short arm from one chromosome of the 14 pair. In meiosis of this type of rearrangement, the father’s normal homologous chromosomes are paired with the mother’s translocated chromosomes and as a result of 3:1 segregation a gamete with one chromosome else was originated that after fertilization resulted in an unbalanced translocation confirming the pure trisomy in the patient.

The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila.

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo-formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.

Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.).

The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development.

Basigin null mutant male mice are sterile and exhibit impaired interactions between germ cells and Sertoli cells

Basigin (BSG) is a multifunctional glycoprotein that plays an important role in male reproduction since male knockout (KO) mice are sterile. The Bsg KO testis lacks elongated spermatids and mature spermatozoa, a phenotype similar to that of alpha-mannosidase IIx (MX) KO mice. MX regulates formation of N-acetylglucosamine (GlcNAc) terminated N-glycans that participate in germ cell-Sertoli cell adhesion. Results showed that Bsg KO spermatocytes displayed normal homologous chromosome synapsis and progression through meiosis. However, only punctate expression of the round spermatid marker SP-10 in the acrosomal granule of germ cells of Bsg KO mice was detected indicating that spermatogenesis in Bsg KO mice was arrested at the early round spermatid stages. We observed a large increase in the number of germ cells undergoing apoptosis in Bsg KO testes. Using lectin blotting, we determined that GlcNAc terminated N-glycans are linked to BSG. GlcNAc terminated N-glycans were significantly reduced in Bsg KO testes. These observations indicate that BSG may act as a germ cell-Sertoli cell attachment molecule. Loss of BSG significantly reduced adhesion between GC-2 and SF7 cells. Moreover, wild type testes showed strong expression of N-cadherin (CDH2) while expression was greatly reduced in the testes of Bsg KO mice. In addition, the integrity of the blood-testis barrier (BTB) was compromised in Bsg KO testes. In conclusion, although some Bsg KO spermatogonia can undergo normal progression to the spermatocyte stage, BSG-mediated germ cell-Sertoli cell interactions appear to be necessary for integrity of the BTB and spermatocyte progression to mature spermatozoa.

Maize meiotic mutants with improper or non-homologous synapsis due to problems in pairing or synaptonemal complex formation

During meiotic prophase homologous chromosomes find each other and pair. Then they synapse, as the linear protein core (axial element or lateral element) of each homologous chromosome is joined together by a transverse central element, forming the tripartite synaptonemal complex (SC). Ten uncloned Zea mays mutants in our collection were surveyed by transmission electron microscopy by making silver-stained spreads of SCs to identify mutants with non-homologous synapsis or improper synapsis. To analyse the mutants further, zyp1, the maize orthologue of the Arabidopsis central element component ZYP1 was cloned and an antibody was made against it. Using antibodies against ZYP1 and the lateral element components AFD1 and ASY1, it was found that most mutants form normal SCs but are defective in pairing. The large number of non-homologous synapsis mutants defective in pairing illustrates that synapsis and pairing can be uncoupled. Of the ten mutants studied, only dsy2 undergoes normal homologous chromosome recognition needed for homologous pairing. The dsy2 mutation fails to maintain the SC. ZYP1 elongation is blocked at zygotene, and only dots of ZYP1 are seen at prophase I. Another mutant, mei*N2415 showed incomplete but homologous synapsis and ASY1 and AFD1 have a normal distribution. Although installation of ZYP1 is initiated at zygotene, its progression is slowed down and not completed by pachytene in some cells and ZYP1 is not retained on pachytene chromosomes. The mutants described here are now available through the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu/).

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier’s embryos- preliminary observations of two robertsonian translocation carrier families

Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

From meiosis to postmeiotic events: Homologous recombination is obligatory but flexible

Sexual reproduction depends on the success of faithful chromosome transmission during meiosis to yield viable gametes. Central to meiosis is the process of recombination between paternal and maternal chromosomes, which boosts the genetic diversity of progeny and ensures normal homologous chromosome segregation. Imperfections in meiotic recombination are the source of de novo germline mutations, abnormal gametes, and infertility. Thus, not surprisingly, cells have developed a variety of mechanisms and tight controls to ensure sufficient and well-distributed recombination events within their genomes, the details of which remain to be fully elucidated. Local and genome-wide studies of normal and genetically engineered cells have uncovered a remarkable stochasticity in the number and positioning of recombination events per chromosome and per cell, which reveals an impressive level of flexibility. In this minireview, we summarize our contemporary understanding of meiotic recombination and its control mechanisms, and address the seemingly paradoxical and poorly understood diversity of recombination sites. Flexibility in the distribution of meiotic recombination events within genomes may reside in regulation at the chromatin level, with histone modifications playing a recently recognized role.

Meiotic chromosome behaviour and Boronia (Rutaceae) genome reorganisation

Meiotic observation of nine genotypes of seven Boronia Sm. species revealed that B. megastigma Bartling (2n = 14), B. molloyae J.R.Drumm. (2n = 16), B. purdieana Diels (2n = 18), B. pinnata Sm. (2n = 22) and B. chartacea P.H.Weston (2n = 32) behaved as diploids during meiosis. B. coerulescens F.Muell. (2n = 72) was a tetraploid. These species had normal homologous chromosome pairing and segregation during meiosis and high pollen viability of 82–99%. Base chromosome numbers for B. megastigma, B. molloyae, B. purdieana, B. pinnata, B. chartacea and B. coerulescens were 7, 8, 9, 11, 16 and 18, respectively. Irregularities were found in meiosis of B. heterophylla F.Muell., which had low pollen viability of 43–46%. Results indicated that chromosome number in B. heterophylla might be the result of dysploid reduction or aneuploid reduction with 'Red' a monosomic, whereas 'Cameo' and 'Moonglow' were double monosomics. Boronia genome appeared volatile and genome repatterning in Boronia is still occurring.

Alpha satellite DNA variant-specific oligoprobes differing by a single base can distinguish chromosome 15 homologs.

The ability to distinguish homologous chromosomes is a powerful cytogenetic tool. However, traditional techniques can only distinguish extreme physical variants and are highly dependent on sample preparation. We have previously reported oligonucleotide probes, specific for human chromosome 17 alpha satellite DNA sequence variants, that distinguish cytogenetically normal homologous chromosomes by FISH. Here we report the development of similar oligoprobes, differing at a single nucleotide position, that not only distinguish homologous chromosomes 15 but can be used to follow the transmission of a chromosome from parents to their offspring. We also identified a novel array-size polymorphism in another family. The alphoid array of one chromosome is quite small and below the detection threshold for our oligoprobes, although it is detectable by conventional FISH probes. This size polymorphism provides an additional FISH-based method for distinguishing homologs. Most importantly, this work illustrates the potential applicability of the technique to the entire human chromosome complement.

Meiotic Chromosome Pairing, Isozyme Analyses and Ferritin Expression in a Red Clover Mutant Capable of Somatic Embryogenesis

Red clover genotypes capable of regenerating plantletsin vitrofrom non-meristem-derived callus are rare. A previous study identified a pair of near isogenic lines which were derived from a single seed but differed in regenerative ability. The callus-derived plants of this clone were highly regenerative when reintroduced to culture whereas the epicotyl-derived plants produced non-regenerative callus cultures. The objective of the present study was to observe meiotic chromosome pairing and to compare isozyme profiles and ferritin gene expression in regenerative and non-regenerative plants and cultures from the clone. Meiotic cells exhibited normal homologous chromosome pairing. Starch gel zymograms from glasshouse-grown regenerative (F49R) and non-regenerative (F49M) plants failed to show somaclonal variation for alcohol dehydrogenase, glutamate dehydrogenase, esterase or peroxidase. Isoelectric focusing of callus cultures from regenerative and non-regenerative plants revealed that regeneration was accompanied by a reduction in staining intensity and numbers of peroxidase bands compared to non-regenerative cultures. A unique cathodic peroxidase band (pI 7.6) was associated with non-regenerative cultures. Ferritin expression was greater in callus than in fresh petiole tissue. Ferritin expression remained high in non-regenerative callus cultures but declined in regenerative cultures as regeneration progressed.

Haplotype-Specific Alphoid Oligonucleotides Can Distinguish Normal Homologous Chromosomes by Fish

The field of molecular cytogenetics is presently hampered by the need for allele- and homologue-specific hybridization probes. Vast stretches of the human genome display a considerable amount of polymorphic variation between individuals. Alpha satellite DNA is a tandemly repeated sequence located at the centromeres of all primate chromosomes and is a rich source of polymorphisms. These DNA variants are phenotypically silent; they are also stable and heritable in Mendelian fashion. We have shown that we can use this genetic diversity to create homologue-specific probes using fluorescence in situ hybridization (FISH) However unlike most classical heteromorphisms, which are detected by banding techniques, our approach does not depend upon detection of gross alterations in chromosome structure, but on the basis of sequence composition. We have constructed highly-specific oligonucleotide probes that are the first reported FISH probes to discriminate between the cytogenetically indistinguishable chromosomes of normal individuals.These sequence-specific probes have been used to follow the transmission of a single chromosome 17 through three generation families, similar to a typical polymorphic marker.

A study of homologous chromosomes using a morphometric approach

A previous study of 100 karyotyped metaphase cells has demonstrated the utility of a graphic arts tool in deriving chromosome measurements for relative length determination. In the present study we utilize this same approach to address the question: "What are the average differences in relative lengths between apparently normal homologous chromosomes?" Normal standards derived from this study will be useful for testing specific hypotheses involving heteromorphic differences between homologs. No such data on normal controls could readily be found in either An International System for Human Cytogenetic Nomenclature (1985) or elsewhere.

Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion.

A high proportion of patients with myelodysplasia show characteristic karyotypic abnormalities in bone marrow cells. The most distinctive of the myelodysplastic syndromes is the 5q- syndrome characterized by refractory anemia, poorly lobulated megakaryocytes, and an interstitial deletion of the long arm of chromosome 5 (5q deletion) as the sole karyotypic abnormality. Recently, several genes encoding hemopoietic growth factors and receptors, comprising the interleukins 3, 4, and 5, macrophage colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, and the receptor for macrophage-colony-stimulating factor [the CSF1R (formerly FMS) gene product], have been localized to the long arm of chromosome 5, and there has been much speculation that deletion of one or more of these genes may be critical to the pathogenesis of the associated myeloid disorders. One candidate gene is CSF1R, which is required for normal proliferation and differentiation of hemopoietic cells of the myeloid lineage. We have carried out a molecular examination of the CSF1R, both on the 5q- chromosome and on the apparently normal homologous chromosome 5, in 10 patients with myelodysplasia and a 5q deletion. We have found, using restriction fragment length polymorphism analysis and gene dosage experiments, that all 10 patients showed deletion of CSF1R; 6 of 10 were hemizygous and 4 of 10 homozygous for CSF1R loss. The homozygous CSF1R loss has been confirmed in 2 patients by an in situ hybridization technique comparing the signal in affected cells to that in control sex-mismatched cells on the same slides. In those patients considered to have homozygous CSF1R loss by DNA experiments the gene was deleted from the 5q chromosome in all cells and from the apparently normal chromosome 5 in a subset of cells. This loss of one CSF1R allele, together with loss in some cells of the remaining allele on the homologous chromosome 5, in patients with myelodysplasia indicates that this is a region of critical gene loss on 5q. The loss of the hemopoietic growth factor receptor gene CSF1R may be important in the pathogenesis of human myeloid leukemia.

Biochemical, ultrastructural and genetic characterization of human acrosomal antigen SP-10

Basigin (BSG) is a multifunctional glycoprotein that plays an important role in male reproduction since male knockout (KO) mice are sterile. The Bsg KO testis lacks elongated spermatids and mature spermatozoa, a phenotype similar to that of alpha-mannosidase IIx (MX) KO mice. MX regulates formation of N-acetylglucosamine (GlcNAc) terminated N-glycans that participate in germ cell-Sertoli cell adhesion. Results showed that Bsg KO spermatocytes displayed normal homologous chromosome synapsis and progression through meiosis. However, only punctate expression of the round spermatid marker SP-10 in the acrosomal granule of germ cells of Bsg KO mice was detected indicating that spermatogenesis in Bsg KO mice was arrested at the early round spermatid stages. We observed a large increase in the number of germ cells undergoing apoptosis in Bsg KO testes. Using lectin blotting, we determined that GlcNAc terminated N-glycans are linked to BSG. GlcNAc terminated N-glycans were significantly reduced in Bsg KO testes. These observations indicate that BSG may act as a germ cell-Sertoli cell attachment molecule. Loss of BSG significantly reduced adhesion between GC-2 and SF7 cells. Moreover, wild type testes showed strong expression of N-cadherin (CDH2) while expression was greatly reduced in the testes of Bsg KO mice. In addition, the integrity of the blood-testis barrier (BTB) was compromised in Bsg KO testes. In conclusion, although some Bsg KO spermatogonia can undergo normal progression to the spermatocyte stage, BSG-mediated germ cell-Sertoli cell interactions appear to be necessary for integrity of the BTB and spermatocyte progression to mature spermatozoa.

Determination of chromosomal area of double minute chromosomes and a homogeneously staining region in HL-60 human leukemia cells by the use of a color image analyzer.

The surface area of chromosomes was measured in two sublines of HL-60 human leukemia cells using a color image analyzer. One subline, HL-60 (dmin), had double minute chromosomes (dmin) and the other line, HL-60 (HSR), showed an abnormal chromosome #8 (8q+) which contained a homogeneously staining region (HSR) within the long arm at band q24. Dmin were not seen in the latter cells. The area of individual chromosomes analyzed from metaphase plates of Giemsa-stained photographic prints proved to be reasonably accurate and reproducible. Moreover, the relative area of each chromosome correlated well both with the relative length of corresponding chromosomes and the relative DNA content. The results indicate that the mean area of a single dmin represents 0.16% of the total genomic area of HL-60 (dmin) cells. The average number of dmin per cell was 8.14, and the total area of all dmin in a representative cell was 1.32% of the entire genome. The 8q+ chromosome had 1.46 times the area of the normal homologous chromosome #8, and the HSR itself had 1.30% of the total chromosomal area of HL-60 (HSR) cells. Thus, perhaps coincidentally, the relative amount of DNA in the HSR in an HL-60 (HSR) cell was similar to the total found in multiple dmin in HL-60 (dmin) cells.

Cis-trans position effect in cancer translocations

Translocations of chromosomes occurring in human cancer cells appear specific to the type of cell from which the cancer arises. To explain the action of these translocations, we propose dual position effect: Position effect of the cis type transforms the cell to malignancy, while position effect of the trans type permits the normal homologous chromosome to express a normal gene product. Thus, the translocation itself has to do with malignant transformation, while the normal homologous chromosome performs its normal function. This dual concept is illustrated by t(8;14) in Burkitt's lymphoma. The concept can be tested with t(2;18) and t(8;22) in lymphoid malignancies and with other translocations and chromosome rearrangements marking human cancer cells.

Position effect in translocation (2;8) in acute lymphocytic leukemia with kappa light chain immunoglobulin expression

We report a correlation between t(2;8) translocation in acute lymphocytic leukemia and kappa light chain immunoglobulin production. Since the kappa chain genes are on chromosome #2, this, as well as data on Burkitt lymphoma, points to the possibility of position effect on the level of gene action. Chromosome #2 in the translocation together with chromosome #8 is concerned with malignancy, while the normal homologous chromosome #2 transcribes kappa chains. This model applies to B cell leukemias and lymphomas with changes in chromosome #2 which will predictably express kappa chains. The model also applies to B-cell malignancies with changes in chromosome #22 which will predictably express lambda chains.

Cytological Study of the Telocentric Chromosome in Seven Monotelotrisomics of Barley

The behavior of the extra telocentric chromosome in seven monotelotrisomics (Triplo 1L, 1S, 2L, 2S, 3S, 4S, 5L) of barley (Hordeum vulgare L.) was studied during meiosis. The telocentric chromosome associated with its normal homologues, forming a heteromorphic trivalent, or remained as a univalent at diakinesis and metaphase I. Tandem V-, ring-and-rod-, and rod-shaped trivalents were observed. An interstitial chiasma was observed frequently between the telocentric and its normal homologous chromosome, suggesting that the telocentric chromosome was associated fairly tightly. The frequency of sporocytes with the trivalent was significantly higher at diakinesis than at metaphase, and it also differed significantly between telotrisomic types. The telocentric chromosome lagged at AI, TI, AII, and TII in varying frequencies, depending on the telotrisomic type. The present study demonstrates that the telocentric chromosomes of barley have a rigid, complete, and stable centromere.