RESUMEN

PURPOSE: We sought to identify autoantigens recognized by antibodies in breast cancer patient sera with potential diagnostic or prognostic significance. EXPERIMENTAL DESIGN: Serum from a female breast cancer patient exhibiting a high titer antinuclear antibody was used to screen a HeLa cDNA expression library, leading to the cloning of a cDNA for the M(r) 32,000 subunit of replication protein A (RPA32). RPA32 expression and localization were assayed in autologous tumor by monoclonal antibody staining. A specific ELISA using recombinant protein was used to screen sera from 801 breast cancer patients and 65 controls. RESULTS: A relationship between anti-replication protein A (RPA) antibodies and the ductal breast carcinoma of the proband was suggested by overexpression and aberrant localization of RPA32 in tumor cells as compared with surrounding normal ductal tissue and by the presence of anti-RPA32 antibodies before the diagnosis. The prevalence of anti-RPA32 antibodies was significantly higher (P < 0.01) among breast cancer patients (87 of 801 patients) than among noncancer controls (0 of 65 controls). Similarly, anti-RPA32 antibodies were present in 4 of 39 patients with intraductal in situ carcinoma. No associations were found between anti-RPA antibodies and survival, occurrence of a second tumor, metastases, or antibodies to p53. Reactivity to RPA32 also was detected in sera from 3 of 47 patients with other cancers. CONCLUSIONS: In view of the central role of RPA in DNA replication, recombination, and repair, we suggest that autoimmunity to RPA32 may reflect molecular changes involved in the process of tumorigenesis. The finding of antibodies to RPA32 before diagnosis and their prevalence in in situ carcinoma suggest that they are potentially useful markers of early disease.

Asunto(s)

Autoanticuerpos/sangre , Autoantígenos/inmunología , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/inmunología , Carcinoma Ductal de Mama/inmunología , Proteínas de Unión al ADN/inmunología , Antígenos de Neoplasias/inmunología , Autoinmunidad , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma in Situ/sangre , Carcinoma in Situ/inmunología , Carcinoma in Situ/patología , Carcinoma Ductal de Mama/sangre , Carcinoma Ductal de Mama/patología , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Femenino , Biblioteca de Genes , Células HeLa , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/patología , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Peso Molecular , Núcleo Familiar , Valores de Referencia , Proteína de Replicación A , Proteína p53 Supresora de Tumor/inmunología

RESUMEN

In recombination-proficient organisms, chiasmata appear to mediate associations between homologs at metaphase of meiosis I. It is less clear how homolog associations are maintained in organisms that lack recombination, such as male Drosophila. In lieu of chiasmata and synaptonemal complexes, there must be molecules that balance poleward forces exerted across homologous centromeres. Here we describe the genetic and cytological characterization of four EMS-induced mutations in teflon (tef), a gene involved in this process in Drosophila melanogaster. All four alleles are male specific and cause meiosis I-specific nondisjunction of the autosomes. They do not measurably perturb sex chromosome segregation, suggesting that there are differences in the genetic control of autosome and sex chromosome segregation in males. Meiotic transmission of univalent chromosomes is unaffected in tef mutants, implicating the tef product in a pairing-dependent process. The segregation of translocations between sex chromosomes and autosomes is altered in tef mutants in a manner that supports this hypothesis. Consistent with these genetic observations, cytological examination of meiotic chromosomes suggests a role of tef in regulating or mediating pairing of autosomal bivalents at meiosis I. We discuss implications of this finding in regard to the evolution of heteromorphic sex chromosomes and the mechanisms that ensure chromosome disjunction in the absence of recombination.

Asunto(s)

Drosophila melanogaster/genética , Genes de Insecto , Meiosis/genética , Alelos , Animales , Cromosomas/genética , Cruzamientos Genéticos , Femenino , Masculino , Mutación , Cromosomas Sexuales/genética , Translocación Genética

RESUMEN

In Drosophila melanogaster, the rDNA loci function in ribosome biogenesis and nucleolar formation and also as sex chromosome pairing sites in male meiosis. These activities are not dependent on the heterochromatic location of the rDNA, because euchromatic transgenes are competent to form nucleoli and restore pairing to rDNA-deficient X chromosomes. These transgene studies, however, do not address requirements for the function of the endogenous rDNA loci within the heterochromatin. Here we describe two chromosome rearrangements that disrupt rDNA functions. Both rearrangements are translocations that cause an extreme bobbed visible phenotype and XY nondisjunction and meiotic drive in males. However, neither rearrangement interacts with a specific Y chromosome, Ymal(+), that induces male sterility in combination with rDNA deletions. Molecular studies show that the translocations are not associated with gross rearrangements of the rDNA repeat arrays. Rather, suppression of the bobbed phenotypes by Y heterochromatin suggests that decreased rDNA function is caused by a chromosomal position effect. While both translocations affect rDNA transcription, only one disrupts meiotic XY pairing, indicating that there are different cis-acting requirements for rDNA transcription and rDNA-mediated meiotic pairing.

Asunto(s)

Segregación Cromosómica/genética , ADN Ribosómico/biosíntesis , Drosophila melanogaster/genética , Cromosomas Sexuales/genética , Transcripción Genética/genética , Alelos , Animales , Rotura Cromosómica/genética , Análisis Mutacional de ADN , ADN Ribosómico/genética , Femenino , Dosificación de Gen , Prueba de Complementación Genética , Heterocromatina , Infertilidad Masculina/genética , Masculino , Meiosis/genética , No Disyunción Genética , Fenotipo , Secuencias Repetitivas de Ácidos Nucleicos , Transgenes , Translocación Genética/genética , Cromosoma X/genética , Cromosoma Y/genética

RESUMEN

In male Drosophila melanogaster, anomalies in sex chromosome pairing at meiosis often lead to complete or partial sperm dysfunction. This observation has led to the suggestion that defects in either the efficiency or configuration of chromosome pairing at metaphase trigger a checkpoint mechanism that leads to the elimination of meiotic products. Here, we discuss this model in consideration of recent observations on the conservation of metaphase checkpoint components in male meiosis, and on the phenotype of new alleles of the male-specific meiotic mutant teflon. Based on these observations, we propose an alternative hypothesis for the cause of sperm dysfunction in cases of chromosomal sterility and drive. We suggest that disruption of the prophase compartmentalization of sex chromatin, rather than abnormal pairing at metaphase, may be the causative defect. Such disruption may occur as a result of perturbations in sex chromosome pairing, or by translocations involving autosomal and sex chromatin. We discuss how this hypothesis may account for previously described examples chromosomal causes of meiotic drive and sterility in Drosophila.

Asunto(s)

Drosophila melanogaster/genética , Meiosis/genética , Animales , Masculino , Cromosoma X , Cromosoma Y

RESUMEN

There are multiple case reports of antinuclear antibodies (ANAs) in patients with malignancies, yet to date there has not been a systematic survey of ANAs in lung cancer. We have previously reported that autoantibodies to collagen antigens resembling those found in the connective tissue diseases are consistently detected in the sera from lung cancer patients. In this work, we looked for the presence of ANAs in the sera from these same patients. Sera from 64 patients with lung cancer and 64 subjects without a history of cancer were retrospectively tested for reactivity on immunoblots of nuclear extracts of HeLa, small cell carcinoma, squamous cell carcinoma, adenocarcinoma, large cell carcinoma of the lung, and of normal lung cells. Associations were sought between the reactivities on immunoblots and lung cancer cell type, diagnosis, and progression-free survival by the method of classification and regression trees (CARTs). Cross-validated CART analyses indicated that reactivities to certain bands in immunoblots are associated with different types of lung cancer. Some of these autoantibodies were associated with a prolonged survival without disease progression. Our data suggest that autoimmunity is often a prominent feature of lung cancer and that molecular characterization of these antigens may lead to the discovery of proteins with diagnostic and prognostic value.

Asunto(s)

Anticuerpos Antinucleares/sangre , Biomarcadores de Tumor/sangre , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos Nucleares , Línea Celular , Supervivencia sin Enfermedad , Femenino , Células HeLa , Humanos , Immunoblotting , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Proteínas Nucleares/inmunología , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia

RESUMEN

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

Asunto(s)

Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Cromosómicas no Histona/biosíntesis , Proteínas Cromosómicas no Histona/química , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Expresión Génica , Humanos , Mamíferos , Mitosis , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia

RESUMEN

Studies of the abnormal oocyte (abo) gene of Drosophila melanogaster have previously been limited to the analysis of a single mutant allele, abnormal oocyte1 (abo1). The abo1 mutation causes a maternal-effect lethality that can be partially rescued zygotically by the abo+ allele and by increasing the dosage of specific regions of heterochromatin denoted ABO. This report describes the properties of abo2, a new P-element-induced allele that allowed us to reexamine the nature of maternal-effect defect. Comparisons of the phenotype of progeny of abo1/abo1 and abo1/abo2 females show that the preblastoderm lethality previously described as a component of the abo mutant maternal effect results from a recessive fertilization defect associated with the abo1 chromosome. We demonstrate here that the abo-induced maternal effect lethality occurs predominately late in embryogenesis after cuticle deposition but before hatching. The phenocritical period for zygotic rescue by heterochromatin coincides with this period of late embryogenesis. We have used the abo2 mutation to map and molecularly clone the gene. We show that the abo gene is located in the 32C cytogenetic interval and identify the putative abo transcript from mRNA isolated from adult females. Using germline transformation, we show that a 9-kb genomic fragment to which the transcript maps, partially fulfills requirement for maternal and zygotic abo+ function.

Asunto(s)

Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Oocitos , Animales , Autorradiografía , Northern Blotting , Southern Blotting , Clonación Molecular , ADN/genética , Drosophila melanogaster/embriología , Femenino , Genes Letales , Mutación de Línea Germinal , Heterocromatina/genética , Infertilidad Femenina/genética , Fenotipo , ARN/genética

RESUMEN

We have isolated cDNAs and raised antibodies corresponding to the human homologs of the S. cerevisiae CDC27 and CDC16 proteins, which are tetratrico peptide repeat (TPR)-containing proteins essential for mitosis in budding yeast. We find that the CDC27Hs and CDC16Hs proteins colocalize to the centrosome at all stages of the mammalian cell cycle, and to the mitotic spindle. Injection of affinity-purified anti-CDC27Hs antibodies into logarithmically growing HeLa cells causes a highly reproducible cell cycle arrest in metaphase with apparently normal spindle structure. We conclude that CDC27 and CDC16 are evolutionarily conserved components of the centrosome and mitotic spindle that control the onset of postmetaphase events during mitosis.

Asunto(s)

Proteínas de Ciclo Celular/aislamiento & purificación , Ciclo Celular/fisiología , Centrosoma/química , Huso Acromático/química , Anafase/efectos de los fármacos , Anafase/fisiología , Anticuerpos/farmacología , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase , Ciclo Celular/efectos de los fármacos , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Metafase/efectos de los fármacos , Metafase/fisiología , Microinyecciones , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia , Ubiquitina-Proteína Ligasas

RESUMEN

The human autoantigen CENP-C has been demonstrated by immunoelectron microscopy to be a component of the inner kinetochore plate. Here we have used antibodies raised against various portions of CENP-C to probe its function in mitosis. We show that nuclear microinjection of anti-CENP-C antibodies during interphase causes a transient arrest at the following metaphase. Injection of the same antibodies after the initiation of prophase, however, does not disrupt mitosis. Correspondingly, indirect immunofluorescence using affinity-purified human anti-CENP-C antibodies reveals that levels of CENP-C staining are reduced at centromeres in cells that were injected during interphase, but appear unaffected in cells which were injected during mitosis. Thus, we suggest that the injected antibodies cause metaphase arrest by reducing the amount of CENP-C at centromeres. Examination of kinetochores in metaphase-arrested cells by electron microscopy reveals that the number of trilaminar structures is reduced. More surprisingly, the few remaining kinetochores in these cells retain a normal trilaminar morphology but are significantly reduced in diameter. In cells arrested for extended periods, these small kinetochores become disrupted and apparently no longer bind microtubules. These observations are consistent with an involvement of CENP-C in kinetochore assembly, and suggest that CENP-C plays a critical role in both establishing and/or maintaining proper kinetochore size and stabilizing microtubule attachments. These findings also support the idea that proper assembly of kinetochores may be monitored by the cell cycle checkpoint preceding the transition to anaphase.

Asunto(s)

Anafase , Centrómero/ultraestructura , Proteínas Cromosómicas no Histona/fisiología , Metafase , Autoantígenos/fisiología , Ciclo Celular , Células HeLa , Humanos , Técnicas In Vitro

RESUMEN

We describe a Drosophila mutation, Double or nothing (Dub), that causes meiotic nondisjunction in a conditional, dominant manner. Previously isolated mutations in Drosophila specifically affect meiosis either in females or males, with the exception of the mei-S332 and ord genes which are required for proper sister-chromatid cohesion. Dub is unusual in that it causes aberrant chromosome segregation almost exclusively in meiosis I in both sexes. In Dub mutant females both nonexchange and exchange chromosomes undergo nondisjunction, but the effect of Dub on nonexchange chromosomes is more pronounced. Dub reduces recombination levels slightly. Multiple nondisjoined chromosomes frequently cosegregate to the same pole. Dub results in nondisjunction of all chromosomes in meiosis I of males, although the levels are lower than in females. When homozygous, Dub is a conditional lethal allele and exhibits phenotypes consistent with cell death.

Asunto(s)

Drosophila melanogaster/genética , Genes de Insecto , Meiosis/genética , Mutación , Alelos , Animales , Muerte Celular/genética , Femenino , Genes Dominantes , Genes Letales , Genes Recesivos , Homocigoto , Masculino , Fenotipo , Recombinación Genética , Caracteres Sexuales

RESUMEN

We have isolated and characterized a set of overlapping cDNA clones that encode the human centromere autoantigen centromere protein C (CENP-C). The identity of these clones has been established using several criteria. First, they were shown to encode a polypeptide that migrates at the expected position for CENP-C on SDS-polyacrylamide gel electrophoresis. Second, we have demonstrated that this polypeptide shares at least two epitopes with human CENP-C. Polyclonal antibodies were raised to fusion proteins encoded by nonoverlapping regions of the cDNA clones. These antibodies were shown to recognize a protein at a position appropriate for CENP-C on immunoblots of human chromosomal proteins. In addition, we used indirect immunofluorescence to demonstrate that these antibodies recognize centromeres of HeLa chromosomes in the expected pattern for CENP-C. Localization of CENP-C by immunoelectron microscopy reveals that this protein is a component of the inner kinetochore plate.

Asunto(s)

Autoantígenos/química , Centrómero/química , Proteínas Cromosómicas no Histona/análisis , Enfermedad de Raynaud/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas/química , Clonación Molecular , ADN/aislamiento & purificación , Células HeLa , Humanos , Datos de Secuencia Molecular , Enfermedad de Raynaud/inmunología

RESUMEN

Recent studies have begun to yield some insight into the structural and regulatory components of centromeres, and new assays have been developed that promise to be of use in advancing our understanding of centromere structure and function. In the budding yeast Saccharomyces cerevisiae new proteins that are required for centromere function have been identified and an in vitro microtubule-binding assay that should assist in dissecting the process of centromere microtubule attachment has been developed. The centromere-specific DNA sequences in the fission yeast Schizosaccharomyces pombe have been identified and partially characterized. In addition, several mammalian centromere proteins have been further characterized, and localization and inhibition studies suggest roles for these proteins in the regulation and assembly of a functional kinetochore.

Asunto(s)

Centrómero/fisiología , Huso Acromático/fisiología , Animales , Autoanticuerpos , Centrómero/ultraestructura , Proteínas Cromosómicas no Histona/fisiología , ADN de Hongos/genética , Humanos , Microtúbulos/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/genética , Schizosaccharomyces/fisiología , Huso Acromático/ultraestructura , Relación Estructura-Actividad

RESUMEN

The euchromatic maternal-effect mutation abnormal oocyte (abo), of Drosophila melanogaster interacts with regions of heterochromatin known as ABO, which reside on the X, Y and second chromosomes. Here, we show that survival of progeny from abo females depends in part upon the maternal dosage of ABO heterochromatin. A comparison was made of the recovery of genotypically identical progeny from abo mothers bearing sex chromosomes of various ABO contents. The results show that the recovery of daughters was decreased if mothers were ABO-/ABO-. However, no decrease was observed if mothers were ABO+/ABO-. In addition, the survival of daughters was greater when they received an ABO-X chromosome from an ABO-/ABO+ mother rather than the father. We suggest that these results reflect a complementation or interaction between the ABO-deficient X and the ABO heterochromatin in the maternal genome. This proposed interaction could occur early in oogenesis in the mother or prior to completion of meiosis I in the fertilized egg. To determine if zygotic dosage of ABO heterochromatin might also be important at very early stages of embryogenesis, we examined the timing of zygotic rescue by paternally donated ABO heterochromatin using a second mutation, paternal loss (pal). Homozygous pal males produce progeny which lose paternally derived chromosomes during the early zygotic divisions. Zygotes that have lost a paternal sex chromosome in a fraction of their nuclei will be mosaic for the amount of ABO heterochromatin. By monitoring the recovery of pal-induced mosaics from abo and abo+ females, we could determine the temporal and spatial requirements for ABO function. Results show that the survival of progeny from the abo maternal-effect lethality was increased if ABO heterochromatin was present prior to the pal-induced loss event. Analysis of mosaic patterns did not reveal a specific lethal focus. We conclude from these results that ABO heterochromatin serves its vital function prior to completion of the early cleavage divisions in progeny of abo mothers.

Asunto(s)

Drosophila melanogaster/genética , Genes Letales/genética , Heterocromatina/fisiología , Animales , Cruzamientos Genéticos , Drosophila melanogaster/embriología , Femenino , Heterocigoto , Masculino , Mosaicismo/genética , Oogénesis/genética , Cromosomas Sexuales/fisiología , Espermatogénesis/genética , Cigoto/crecimiento & desarrollo