Chicken Nuclei Research Articles

The Use of Primed in situ Synthesis (PRINS) to Analyze Nucleolar Organizer Regions (NORs) and Telomeric DNA Sequences in the Domestic Chicken Genome

One of the most often analyzed avian genomes is the domestic chicken genome (Gallus domesticus) whose diploid number is 2n = 78. In the chicken karyotype, similarly to other birds, there is a group of microchromosomes for which the determination of morphology and banding pattern is impossible using classic cytogenetics methods. The aim of this study was to evaluate telomeric and rDNA repetitive sequences in the chicken genome by the PRINS technique as an alternative method to fluorescence in situ hybridization. This is the first report on the application of the PRINS method to locate these repetitive sequences in the chicken nuclei and metaphase chromosomes.

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

Appearance and origin of snRNP antigens in chick erythrocyte nuclei reactivated in heterokaryons

Fusion of terminally differentiated chick erythrocytes (CE) with transcriptionally active rat myoblasts results in heterokaryons in which the CE nuclei undergo reactivation of RNA synthesis and splicing. In order to analyze the transport and assembly of small nuclear ribonucleoprotein (snRNP) particles and larger molecular complexes engaged in RNA processing, we have examined CE nuclei in heterokaryons for the presence of four U snRNP-related nuclear antigens (Sm, 70,000 Mr, F78 and M3G-cap) and for one antigen (La), associated with RNA polymerase III transcripts. Inactive erythrocyte nuclei showed low levels of Sm and F78 antigens, but the other antigens were undetectable. Immediately after fusion, the fluorescence of the pre-existing chicken Sm antigen was detected in the CEn, and then the intensity of the signal increased rapidly during reactivation. The other antigens appeared more slowly, reaching full intensity at different time points after fusion. Blocking of chick transcription did not block the appearance of Sm, 70,000 Mr, cap and La antigens but did effectively inhibit the appearance of the F78 antigen. It has previously been demonstrated that the structure recognized by this monoclonal antibody is physically associated with functional splicing complexes. Blocking of translation in heterokaryons abolished uptake of snRNPs into the chicken nuclei. Taken together, the results indicate that rat snRNP complexes were imported into the chick nuclei after assembly in the cytoplasm. For all the studied antigens, except F78, this translocation was independent of chick RNA synthesis. The appearance of the F78 antigen was totally dependent on expression of chicken genes.

Differences in nuclear thyroid hormone receptors among species

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3′-triiodo- l-thyronine (T 3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T 3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 ± 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T 3 at 22° for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 ± 1.47 × 10 5 M −1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 ± 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T 3-receptor complex using trypsin α-chymotrypsin, elastase, and papain produced distinct T 3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.

A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones.

We have detected and begun to characterize a 17-kD centromere-specific protein, CENP-A (Earnshaw, W. C., and N. Rothfield, 1985, Chromosoma., 91:313-321). Sera from several humans with CREST scleroderma autoimmune disease (CREST: calcinosis, Raynaud's phenomenon, esophageal dsymotility, sclerodactyly, and telangiectasia) bind this protein in immunoblot assays of HeLa whole cell or nuclear extracts. We have affinity purified the anti-17-kD centromere protein (anti-CENP-A) specific antibodies from immunoblots of HeLa nuclear protein. The antibodies react with epitopes present on CENP-A derived from humans but apparently do not recognize specific epitopes in either rat or chicken nuclei. Only human nuclear protein is CENP-A positive by immunoblot. Furthermore, human cells show localization of anti-CENP-A antibody to centromeres by immunofluorescence microscopy, whereas rat cells do not. On extraction from the nucleus, CENP-A copurifies with core histones and with nucleosome core particles. We conclude that this centromere-specific protein is a histone-like component of chromatin. The data suggest that CENP-A functions as a centromere-specific core histone.

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.

Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.

Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.

Cross-reactivity of antilymphocyte and antinuclear antibodies in systemic lupus erythematosus

Both antinuclear antibody (ANA) and antilymphocyte antibody (ALA) are prevalent in systemic lupus erythematosus (SLE). This report presents data supporting cross-reactivity between these two antibodies. Differential absorption of SLE sera showed that chicken erythrocyte nuclei absorbed IgM ALA while intact human and chicken erythrocytes had no effect. RNase digestion of chicken nuclei fragments partially removed the ALA absorbing effect. Both IgM and IgG ANA were also absorbed by the chicken nuclei. Absorptions with intact human lymphocytes removed ALA activity and absorption with human mononuclear cells removed ANA activity. ALA and ANA were both found in PBS eluates from absorbed chicken nuclei, demonstrating specificity of nuclear absorption.

Hydrolysis of histones by horse urinary kallikreins.

Horse urinary kallikrein was shown to hydrolyze histones from calf thymus and chicken nuclei erythrocytes. The hydrolysis is inhibited by benzamidine and hyposulfite but not by soy-bean trypsin inhibitor; horse plasma kallikrein also produces this hydrolysis.

Accidental persistent infection of cell lines by Newcastle disease virus, showing three unusual features--defective neuraminidase, temperature sensitivity and intranuclear inclusions.

A persistent, defective infection by an unknown strain of Newcastle disease virus (NDV) appeared accidentally in established lines of pig, ox and sheep kidney cells. Virus particles released from the persistently infected cells were not infectious and were deficient in neuraminidase activity. Synthesis of some of the virus-specified proteins in the persistently infected cells was temperature-sensitive. Co-cultivation of mixed populations of carrier cells and healthy chick embryo cells induced cell fusion with the formation of multinucleate heterokaryons and intra-nuclear inclusions. The development of inclusions in the chicken nuclei was not accompanied by 'rescue' of infectious NDV.

Ultrastructural study of heterokaryons from rous rat sarcoma cells and normal chickens cells.

Rous virus transformed rat cells containing the Rous virus genome but lacking the ability to produce Rous virus particles have been fused with chicken erythrocytes, chicken lymphocytes or chicken fibroblasts using Sendai virus. Heterokaryotic cells with both rat sarcoma cell nuclei and chicken cell nuclei have been studied in the electron microscope. Shortly after the fusion, a series of changes in the chicken nuclei are demonstrable, which as seen in the electron microscope imply a “reactivation” and production of intranuclear RNA‐containing structures as well as a development of nucleoli. In parallel, the chicken nucleus increases in size and the chromatin is transformed from condensed aggregates into a finely dispersed material. In spite of all evidence of a protein synthesis in the heterokaryotic Rous sarcoma cell directed from the chicken nucleus there is no formation of virus particles in cells with Rous rat sarcoma cells and chicken erythrocytes or chicken lymphocytes. In cultures with Rous rat sarcoma cell—chicken fibroblast heterokaryons a regular synthesis was seen. It seems apparent that the chicken fibroblast contains some factor, whether genetically coded or not, that is required if a production of complete Rous virus particles is to be achieved.