RESUMO
Most individuals with cat eye syndrome (CES) have a supernumerary bisatellited chromosome which, on the basis of cytogenetic evidence, has been reported to originate from either chromosome 13 or 22. To resolve this question, a single-copy DNA probe, D22S9, was isolated and localized to 22q11 by in situ hybridization to metaphase chromosomes. The number of copies of this sequence was determined in CES patients by means of Southern blots and densitometry analysis of autoradiographs. In patients with the supernumerary chromosome, four copies were found, whereas in one patient with a duplication of part of chromosome 22, there were three copies. Therefore, the syndrome results from the presence of either three or four copies of DNA sequences from 22q11; there is no evidence that sequences from other chromosomes are involved. This work demonstrates how DNA sequence dosage analysis can be used to study genetic disorders that are not readily amenable to standard cytogenetic analysis.
Assuntos
Anormalidades Múltiplas/genética , Aberrações Cromossômicas/genética , Coloboma/genética , Transtornos Cromossômicos , Cromossomos Humanos 13-15 , Cromossomos Humanos 21-22 e Y , DNA/genética , Humanos , Hibridização de Ácido Nucleico , SíndromeRESUMO
As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antibody to the coiled body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 coiled body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.
Assuntos
Núcleo Celular/ultraestrutura , Estradiol/farmacologia , Fígado/efeitos dos fármacos , Proteínas Nucleares/análise , Organelas/efeitos dos fármacos , Processamento Pós-Transcricional do RNA , Animais , Nucléolo Celular/ultraestrutura , Galinhas , Fígado/ultraestrutura , Masculino , Microscopia de Fluorescência , Organelas/química , Organelas/ultraestrutura , Precursores de RNA/metabolismoRESUMO
Steroid and similar hormones comprise the broadest class of gene regulatory agents known, spanning vertebrates through the lower animals, and even fungi. Not unexpectedly, therefore, steroid receptors belong to an evolutionarily highly conserved family of proteins. After complexing with their cognate ligands, receptors interact with hormone response elements on target genes and modulate transcription. These actions are multifaceted and only partly understood, and include large-scale changes in the structure and molecular composition of the affected cell nuclei. This chapter examines steroid hormone action and the resultant nuclear remodeling from the following perspectives: (1) Where are the receptors located? (2) Which nuclear domains are most affected? (3) Are there extended or permanent nuclear changes? (4) What is the role of coiled bodies and similar structures in this regard? To address these and related questions, information is drawn from several sources, including vertebrates, insects, and malignant tissues. Entirely new data are presented as well as a review of the literature.
Assuntos
Núcleo Celular/fisiologia , Hormônios/fisiologia , Esteroides/fisiologia , Animais , Regulação da Expressão Gênica/fisiologia , HumanosRESUMO
The hormonal induction of vitellogenesis in insects and in oviparous vertebrates are prime models of gene regulation in eukaryotes. In vertebrates the process is under estrogenic control and normally confined to females, although males can be artificially induced. In locust in contrast, juvenile hormone (JH) is central to fat body development in both males and females, yet the response is strongly sex limited not only for vitellogenin production but also in terms of total protein, DNA and RNA synthesis and nuclear ploidy levels. To differentiate further possible sex and/or JH related developmental aspects in locusts, large-scale nuclear events were examined during normal adult maturation and in animals treated with antiallatropins and JH analogs. Fat body nuclei undergo extensive restructuring during normal development in both sexes. This included progressive nuclear enlargement, accompanied by extensive proliferation of nuclear matrix components and elaboration of complex inclusion bodies (NB). The isolated protein matrix was unusually complex relative to similar structures from vertebrates and the NB were firmly anchored to it. Although matrix proteins were qualitatively similar to those from other sources, as assessed by SDS polyacrylamide gel electrophoresis, several major matrix polypeptides, including lamins A and B, and components greater than 150 kD, fluctuated quantitatively during development and in concert with nuclear enlargement. The number and morphology of the NB were unrelated to sex, but increased in direct proportion to absolute nuclear volumes. All changes were more pronounced in females, where higher ploidy levels, larger nuclei and correspondingly more internal matrix elements occurred. Suppression of JH production by precocene prevented all foregoing nuclear changes, but re-exposure to methoprene rapidly induced normal development. The results are compared to analogous nuclear changes in steroid responsive vertebrate tissues.
Assuntos
Tecido Adiposo/ultraestrutura , Corpo Adiposo/ultraestrutura , Gafanhotos/anatomia & histologia , Hormônios Juvenis/fisiologia , Animais , Benzopiranos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/crescimento & desenvolvimento , Feminino , Gafanhotos/crescimento & desenvolvimento , Masculino , Metoprene/farmacologia , Microscopia Eletrônica , Maturidade SexualRESUMO
Hepatocyte nuclei in several species of vertebrates were examined, to establish the frequency of polyploidy and related parameters along evolutionary lines. Nuclei were compared in terms of volume, DNA content, ploidy ranges and internal organization. Several trends emerged. When present, heterochromatin occupied 20--25% of nuclear profile areas, irrespective of nuclear volume and poloidy; the volume of heterochromatin, however, increased in direct proportion to ploidy level. Regardless of internal organization, ploidy and species, a direct correlation emerged between the volumes of nuclei and their absolute DNA content. Results are discussed in terms of structural and genic DNA.
Assuntos
Evolução Biológica , Fígado/ultraestrutura , Poliploidia , Animais , Nucléolo Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Galinhas , Cromatina/ultraestrutura , DNA/metabolismo , Peixes , Heterocromatina/ultraestrutura , Camundongos , Microscopia Eletrônica , Ratos , TartarugasRESUMO
Hepatocyte nuclei from young (3--5 week), mature (8--12 week), and aged (over 32 weeks) rats were isolated and characterized by flow cytometry. Nuclei were bulk separated into diploid (2C), tetraploid (4C), and octoploid (8C) enriched fractions on sucrose gradients. Total, 0.35 M NaCl soluble, and residual proteins were prepared from all nuclear stages and examined by one- and two-dimensional gel electrophoresis. Within limits of sensitivity of these techniques, the following general features emerged. (a) A majority of proteins visualized were common to and present in similar relative quantities in nuclei from all age and ploidy groups. (b) A relatively higher proportion of nonhistone proteins (NHP) were saline-soluble in 2C nuclei from young rats than at any subsequent stage of development. (c) Several age-related and to a lesser extent ploidy-related fluctuations in pattern among the NHP were evident. These reflected primarily differences in solubility rather than major quantitative changes among individual proteins. (d) Exceptions to the foregoing included a group of high molecular weight components (greater than 100 000), a major and a minor component between 45 000 and 50 000, and a heterogeneous group of proteins in 2C nuclei from very young animals. There were no obvious differences among the histones, although these proteins were not examined in detail. The complex pattern of changes observed are discussed in terms of known aspects of hepatocyte differentiation and are related to possible changes in nucleoplasmic, nuclear matrix and Hn-RNP associated proteins.
Assuntos
Envelhecimento , Fígado/metabolismo , Nucleoproteínas/metabolismo , Ploidias , Animais , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Histonas/metabolismo , Ratos , Ratos Endogâmicos , Ribonucleoproteínas/metabolismoRESUMO
Clarification of large-scale organization in cell nuclei is central to a full understanding of the mechanisms which underlie replication, transcription, and interaction with the cytoplasmic compartment. Many drugs and natural biological agents affect such processes directly, through inhibitory or stimulatory action, and often reveal structural and functional aspects in nuclei not otherwise apparent. There is increasing evidence that the pore complex - lamina of the nuclear matrix is a primary structural element during interphase, which is pivotal to most nuclear processes. This communication reviews evidence based on studies with drugs, inhibitors, and natural biological processes, that the nuclear matrix concept is indeed valid. It is further shown that despite diversity in mode of action and affected target cells, many compounds induce related or complementary changes in nuclei and that such uniformity in pattern is attributable to the nuclear matrix. A general model of the functional organization of the nucleus is presented, to account for the perturbations observed after mass interference with either transcription, replication, or protein synthesis.
Assuntos
Núcleo Celular/ultraestrutura , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Linfócitos/fisiologia , Linfócitos/ultraestrutura , Microscopia Eletrônica , Modelos Biológicos , Matriz Nuclear/fisiologia , Matriz Nuclear/ultraestrutura , Proteínas Nucleares/metabolismo , Transcrição GênicaRESUMO
The estrogenic regulation of vitellogenesis in chicken liver provides an unique perspective on cellular reprogramming because males can be induced experimentally. Transient exposure to estradiol (ES) completely alters established patterns of gene expression in rooster hepatocytes within 6 hrs., and triggers major structural and compositional changes in cell nuclei by 24 hrs. Concurrently, the total protein content of nuclei increases nearly 50% and the relative proportion of protein within the nucleoplasmic, chromatin and residual compartments, shifts markedly. These bulk quantitative changes in nuclear composition are accompanied by marked alterations in 2-D electrophoretic patterns of cytoplasmic, nuclear and nuclear matrix polypeptides. Although most individual proteins remain unidentified, several components clearly overlap both the cytoplasmic and nuclear compartments. Reciprocal changes in the 2-D patterns are also evident after ES stimulation, with progressive decline in some and relative increase in other proteins. Among known species, the lamins (La and Lb) decrease in prominence after hormone, while RNP-associated polypeptides become increasingly pronounced in the residual matrix fraction. The results are discussed in relation to other systems where large-scale nuclear reprogramming is known to occur.
Assuntos
Fígado/química , Proteínas Nucleares/análise , Vitelogeninas/biossíntese , Animais , Proteínas Sanguíneas/análise , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Células Cultivadas , Galinhas , Estradiol/farmacologia , Laminas , Fígado/efeitos dos fármacos , Masculino , Ribonucleoproteínas/análiseRESUMO
The target cells of most steroid hormones contain prominent structures termed nuclear bodies (NB). Estrogen (ES) responsive tissues have very distinctive NB, whose development has been associated with both the level of ES receptors and their retention times in nuclei. It has been suggested that these NB may be large-scale ES binding centers at transcriptionally hyperstimulated sites. We examined the formation of NB in ES-stimulated rooster liver, after primary induction of vitellogenesis and during subsequent decay of this response. Distinct NB appear 6 h postinduction, initially as compact spheroids with ring-like profiles and later on as more complex morphotypes. Nuclei average 15-17 NB each at peak induction (24-48 h), but this declines again gradually as the hormonal response abates. This transient pattern is paralleled closely by the [3H]ES binding capacity of nuclei in vitro and the calculated number of specific hormone receptors present. There is no direct numerical correspondence, however, between the NB and the ES receptors, either on a one-to-one basis or as some simple ratio. Hence any links between ES binding activity in nuclei and the NB must be both complex and indirect. Nuclear bodies were isolated following lysis of nuclei by sonication and separation of subfractions on discontinuous sucrose gradients. The resultant NB-enriched fraction consisted primarily of protein, with traces of DNA and RNA. Analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a complex pattern of polypeptides, some apparently unique and probably NB-derived.
Assuntos
Núcleo Celular/ultraestrutura , Estradiol/farmacologia , Fígado/ultraestrutura , Animais , Fracionamento Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Galinhas , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microscopia Eletrônica , Peso Molecular , Proteínas Nucleares/isolamento & purificação , Receptores de Estradiol/metabolismo , Valores de ReferênciaRESUMO
Alterations in the structure and molecular composition of avian hepatocyte nuclei were compared following administration in vivo of lethal and sub-lethal doses of alpha-amanitin. This toxin interferes with extranucleolar transcription by direct inhibition of RNA polymerase II activity. the resultant effects include: extensive condensation of chromatin, displacement of nucleoplasmic contents and fragmentation of nucleoli. Changes in nuclear morphology were quantitated by stereometry and related to variations in RNA and residual, non-histone proteins (NHP). Gross alterations in nuclear structure and depletion of RNA and NHP levels were of similar magnitude with both doses of amanitin. The effects were fully reversible, however, with a minimal dose but terminal with a lethal dose. DNA and histone protein levels remained unchanged at all stages. These results imply that the process of transciption may itself keep and/or maintain chromatin in a dispersed state, and that in the absence of transcription chromatin naturally condenses. Modification of nuclear proteins may be necessary only to maintain chromatin compacted permanently or for extended periods of time. A model of nuclear organization is proposed to incorporate these considerations and to identify the probable location of the nuclear matrix in situ.
Assuntos
Amanitinas/farmacologia , Núcleo Celular/efeitos dos fármacos , Fígado/citologia , Animais , Fracionamento Celular , Galinhas , RNA Polimerases Dirigidas por DNA/metabolismo , Fígado/ultraestrutura , Ácidos Nucleicos/análise , Proteínas/análiseRESUMO
The process of erythropoiesis is characterized by several distinctive features which render it a very useful model of cell differentiation. Mature erythrocytes arise from stem cells in a series of intermediate stages which are fairly well defined both on morphological and on biochemical grounds. During this development, the erythrocytes genome is gradually inactivated and the cell becomes geared to the production of primarily one gene product, hemoglobin. Recently, erythropoiesis has been closely studied in avian species since it has become technically possible to fractionate the blood of anemic birds into high-yield populations of young, developing and mature red cells. Attention has focused on patterns of RNA synthesis including globin m-RNA, in relation to cytoplasmic constitutents becoming modified for reduced activity. From the point of view of gene regulation, erythrocyte development is especially interesting in non-mammals, where in contrast to mammals, even fully mature red cells retain their nuclei. These erythrocytes rank among the most extreme examples of cell specialization and gene repression known. The nuclei of avian erythrocytes and others, contain a tissue-specific histone protein in addition to the more usual complement of vertebrate histone. This histone (H5, V, F2c) has been extensively investigated with a view to linking its presence to structural and molecular changes involved in the condensation and repression of red cell nuclei. The evidence to dat suggests that H5, in conjunction with tissue-specific changes in non-histone proteins, may be responsible for keeping the genomes of nucleated erythrocytes permanently inactive.
Assuntos
Eritrócitos/citologia , Eritropoese , Animais , Aves/sangue , Diferenciação Celular , DNA/biossíntese , Eritroblastos/citologia , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Eritrócitos/fisiologia , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/fisiologia , Histonas/fisiologia , Mitose , Nucleoproteínas/fisiologia , RNA/biossíntese , RNA Mensageiro/biossíntese , Reticulócitos/citologiaRESUMO
Nuclear bodies (NBs) were first described in detail some 30 years ago, by conventional electron microscopy, as prominent interchromatin structures found primarily in the nuclei of malignant or hyperstimulated animal cells. Subsequent studies have shown that NBs are ubiquitous organelles, but they are numerically and morphologically quite varied. With the recent discovery of human autoantibodies against several key nuclear antigens present in some NBs, these structures are once again the subject of much attention. At least one class of NBs, coiled bodies, has been shown to be nucleolus-derived and to contain not only nucleolus-associated antigens, but also many of the snRNP components involved in pre-mRNA splicing. These data suggest that coiled bodies, and perhaps other NBs as well, are multifunctional and may be involved in the processing or transport of both pre-mRNA and pre-rRNA. Further evidence is provided showing that NBs constitute distinct nuclear domains whose functional significance is just now emerging.
Assuntos
Núcleo Celular/ultraestrutura , Organelas/ultraestrutura , Animais , Humanos , Microscopia Eletrônica , Organelas/metabolismoRESUMO
Primary suspension cultures of hepatocytes from adult roosters were prepared by collagenase perfusion. The cells remained viable for better than 12h as assessed by vital dye exclusion, electron microscopy and protein synthesis. In addition to the spectrum of proteins observed in control cultures, hepatocytes from animals pretreated with 17 beta-oestradiol synthesized vitellogenin, low density lipoprotein and several other unidentified peptides. Vg production, as determined by direct immunoprecipitation, was maintained at levels comparable to those observed in vivo with oestrogen treated roosters. Attempts to attain secondary induction of vitellogenesis in vitro, however, using cells from animals in which the primary response had subsided, were not successful. Nonetheless, the results show as a first step, that suspension cultures of liver cells from adult birds can be prepared which remain viable and morphologically differentiated for an extended period.
Assuntos
Galinhas/anatomia & histologia , Estradiol/farmacologia , Lipoproteínas/biossíntese , Fígado/citologia , Vitelogeninas/biossíntese , Animais , Separação Celular , Sobrevivência Celular , Células Cultivadas , Lipoproteínas VLDL/biossíntese , Fígado/metabolismo , Masculino , Biossíntese de ProteínasRESUMO
Normal and denatured polytene chromosomes from Drosophila were compared by light, transmission (TEM) and scanning (SEM) electron microscopy, under different conditions of pretreatment, fixation and specimen preparation. Apart from variations in contrast, acid-alcohol squashes of normal chromosomes were quite comparable in terms of resolution and image quality, regardless of mode of visualization, i.e. stained, unstained, phase contrast, etc. These chromosomes do not appear markedly different from the unfixed or "native" chromosomes isolated by other workers. Thus at the light microscope level at least, any soluble components extracted by acid-alcohol fixation seem to play minor structural roles only. SEM examination of similar squash preparations provided useful topographic views of selected chromosome regions, particularly in favourable spreads where metal coating and tilt angles were optimized. Information on the internal organization of polytene chromosomes was sought by subjecting isolated preparations to alkali-urea denaturation. Under these conditions, chromosomes gradually extend laterally to give rise to lampbrush-like structures, remarkably similar to the meiotic chromosomes of amphibian oocytes. This effect is at least partially reversed by return of alkali-urea treated preparations to physiological Ringer solution, where chromosomes contract again and near-normal banding patterns are re-established. It is suggested that apart from bulk differences due to polyteny, the basic structure of polytene chromosomes and true lampbrush chromosomes may be rather similar.
Assuntos
Cromossomos/ultraestrutura , Acetatos , Animais , Cromatina/ultraestrutura , Bandeamento Cromossômico , Técnicas Citológicas , Drosophila melanogaster , Microscopia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Coloração e Rotulagem , UreiaRESUMO
The estrogenic induction of vitellogenesis in avian and amphibian liver has been well characterized at the target gene level. Comparatively little however, is known about cognate nuclear events associated with the response, particularly those involving large-scale structural changes and the non-histone proteins (NHP). We have examined these aspects further in primary stimulated roosters. In the first 24 hr post induction with estradiol, hepatocyte nuclei enlarged by 50% and exhibited sharp rises in total protein and RNA content. In particular, the mass of residual NHP rose about 40%. Extensive internal reorganization was evident, including partial disaggregation of chromatin, proliferation of interchromatin components and de novo appearance of prominent "nuclear bodies". These changes were accompanied by quantitative fluctuations in nucleoplasmic and several matrix fraction proteins. A marked relative decrease was evident in all three lamins, as well as approximately 75 and approximately 175 kD proteins. Hn-RNP-associated polypeptides however, and various unidentified components became much more prominent. By 24 hr, cells were fully differentiated for bulk export of vitellogenin and low density lipoproteins. All changes persisted for several days before gradually regressing to normal over a 2-4 week period. Many key nuclear modifications, however, did not regress fully, including persistent enlargement, elevated NHP content and modified matrix fraction proteins. Collectively, these may reflect part of the "memory" effect, commonly observed in steroid target tissues, whereby a second, more pronounced response can be triggered long after primary induction has subsided.
Assuntos
Núcleo Celular/ultraestrutura , Estradiol/farmacologia , Fígado/ultraestrutura , Nucleoproteínas/metabolismo , Vitelogênese/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Galinhas , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Heterocromatina/metabolismo , Lipoproteínas LDL/sangue , Fígado/metabolismo , Masculino , Microscopia Eletrônica , RNA/metabolismo , Vitelogeninas/sangueRESUMO
Transient blockage of transcription in chick liver by alpha-amanitin results in a temporary clearing of nucleoplasmic regions in affected nuclei and concurrent exposure in situ of a discrete interchromatin framework. This is accompanied by sharp decreases in total nuclear RNA (approximately 28%) and nonhistone protein content (approximately 18%). Electrophoretic analysis of matrix fraction proteins from such highly denuded nuclei shows a marked relative enrichment in actin and RNP-derived polypeptides. These observations suggest that actin is a real constituent of the interchromatin region and supports various reports that this protein may have a basic structural role in nuclei.
Assuntos
Actinas/análise , Cromatina/análise , Fígado/ultraestrutura , Amanitinas/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Galinhas , Proteínas Cromossômicas não Histona/metabolismo , Eletroforese , Microscopia Eletrônica , RNA/metabolismo , Ribonucleoproteínas/análise , Transcrição Gênica/efeitos dos fármacosRESUMO
A major component of nuclear change in concanavalin-A-stimulated bovine lymphocytes is a severalfold increase in interchromatinic volume, which coincides with nuclear swelling and extensive structural remodelling. Large-scale ultrastructural changes in isolated nuclei and nuclear matrices (NM) reflect those occurring within nuclei in situ during mitogenesis. While nonchromatinic nuclear material embedded within nuclease- and salt-extracted whole cells closely resembled in situ interchromatinic matrices, large NM isolated in solution shrank after chromatin was extracted. Numerous perinuclear filaments persisted throughout NM isolation and cytoskeletal proteins were identified in two-dimensional (2-D) gels of such preparations. Taken together these data indicated that the lymphocyte cytoskeleton is likely continuous with the nuclear matrix and could play a role in maintaining nuclear organization. A wide range of lymphocyte NM proteins were resolved in 2-D gels. Significant changes in protein composition coincided with nuclear structural remodelling. Lamin B was prominent at each stage of nuclear development, whereas lamins A and C were only found in stimulated lymphocyte matrices. Lymphoblast NM contained more large basic proteins. Progressively increasing polypeptide complexity of these NM arose by de novo protein synthesis and posttranslational modifications throughout concanavalin A stimulation. NM from stimulated lymphocytes also contained more ribonucleoproteins, possibly indicating the presence of significant amounts of transcriptional material.
Assuntos
Núcleo Celular/metabolismo , Concanavalina A/farmacologia , Ativação Linfocitária , Linfócitos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Bovinos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Microscopia Eletrônica , Proteínas Nucleares/isolamento & purificaçãoRESUMO
The regenerating blood of geese injected with phenylhydrazine was subjected to large scale, zonal centrifugation through density gradients of Ficoll. In this way, erythroid cells were fractionated according to their respective stages of development. Highly enriched fractions were obtained, containing cells that were well preserved as assessed by both light and electron microscopy. The separated cells exhibited ribosome density and nucleic acid and protein staining patterns typically associated with erythrocyte differentiation. Morphometric analysis of nuclei indicated that despite an apparent net increase in the amount of compact chromatin during development, comparatively little difference existed between the volumes of condensed chromatin present in immature and mature cells. Instead, there was a three fold decrease in nuclear volume between young erythroblasts and reticulocytes, coupled with a concomitant decrease in the volume occupied by dispersed chromatin, RNP and nucleoli. These observations are discussed in relation to molecular changes associated with nuclear differentiation in erythroid cells.
Assuntos
Eritroblastos/ultraestrutura , Eritrócitos/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Separação Celular/métodos , Centrifugação Zonal , Cromatina/análise , Eritropoese , Gansos , Microscopia EletrônicaRESUMO
We constructed a library in lambda L47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag+/Cs2SO4 gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of alpha-R1-DNA and the X specific alpha-satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived alpha-satellite sequences analogous to other chromosome-specific satellite sequences described previously.
Assuntos
Cromossomos Humanos Par 22/análise , DNA Satélite/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Animais , Cricetinae , DNA Recombinante/análise , Humanos , Células Híbridas/análise , Mapeamento de NucleotídeosRESUMO
Multiple endocrine neoplasis type 2A (MEN2A) is one of several kinds of cancers that appear to be inherited in an autosomally dominant fashion. We have assigned the MEN2A locus to chromosome 10 by linkage with a new DNA marker (D10S5). The linkage led us to investigate other chromosome 10 markers and demonstrate linkage between the disease locus and the interstitial retinol-binding protein (IRBP) gene. The D10S5 locus was sublocalized to 10q21.1 by hybridization in situ and the IRBP gene to p11.2----q11.2 with a secondary site at q24----q25. The linkages were established using 292 members of five families, three different restriction fragment length polymorphisms (RFLPs) at D10S5 and two RFLPs recognized by the IRBP probe. The recombination frequencies from pairwise linkage analysis between the disease and two marker loci D10S5 and IRBP were 0.19 and 0.11, with maximum lod scores of 3.6 and 8.0 respectively. Ordering of the three loci by multipoint analysis placed the IRBP gene approximately midway between the disease and D10S5 loci.