RESUMEN

The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3' MAR of Emu stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is no mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5' and 3' MARs of Emu and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Emu core.

Asunto(s)

RESUMEN

Here we compare the regulation of V(D)J recombination in the fibroblast cell line L4 and the pre-B cell line 38B9. The former has been rendered recombination-competent by stable transfection of a genomic fragment comprising the recombination activating genes, RAG-1 and RAG-2, along with some of their flanking sequences. We show that V(D)J recombination is similarly regulated in these two cell lines. Activating signals are transmitted through the protein kinase A (PKA) pathway, and inhibitory signals through the protein kinase C (PKC) and the calcium signalling pathways. In both cell lines, recombinational activity reflects steady state levels of mRNA transcribed from the RAG-1 and RAG-2 genes. This suggests that transcription of the RAG genes is a major determinant regulating V(D)J recombination. A comparison of RAG-1 and RAG-2 mRNA levels within each cell line reveals almost identical response patterns indicating that RAG-1 and RAG-2 transcription is coordinately regulated. Together, these results imply that the RAG-containing fragment in L4 fibroblasts carries most, if not all, control regions regulating the transcription of the RAG-1 and RAG-2 genes.

Asunto(s)

RESUMEN

DNA-fingerprinting has become, during the last five years, an important method of genetic analysis in medicine, veterinary medicine and other disciplines. The power of this technique, especially for genetic linkage analysis, may be enhanced in humans by using the two dimensional DNA-fingerprinting method. Here we show that this procedure can successfully be applied to different animal species, e.g. pig, dog and mouse. Optimal conditions, however, have to be determined for each species tested. With the use of marker systems as well as computer programs it will be possible to evaluate complex two-dimensional spot patterns in a short time and with high reliability.

Asunto(s)

RESUMEN

We have previously isolated from bull testis three proteins of molecular mass 31, 33, and 58 kDa that we have tentatively characterized as high affinity Z-DNA-binding proteins. This inference was based on their preferential binding to brominated poly(dG-dC).poly(dG-dC) in Z-form as opposed to the unbrominated polynucleotide in B-form (Gut, S. H., Bischoff, M., Hobi, R., and Kuenzle, C. C. (1987) Nucleic Acids Res. 15, 9691-9705). By partial amino acid sequencing we have provisionally identified the 31- and 33-kDa proteins as members of the high mobility group 2 and 1 protein families, respectively, whereas the 58-kDa protein has so far remained unidentified (Christen, Th., Bischoff, M., Hobi, R., and Kuenzle, C. C. (1990) FEBS Lett. 267, 139-141). In the present study, we have critically reassessed the binding specificity of these three proteins by using more natural Z- and B-DNA ligands. As such we chose supercoiled and relaxed DNA minicircles containing a d(CG)7 insert in the Z- and B-conformation, respectively. Filter binding tests and gel retardation assays performed with these ligands showed that the three testis proteins either do not discriminate between Z- and B-DNA (31- and 33-kDa proteins) or even have a preference for B-DNA (58-kDa protein). Therefore, we question the validity of using brominated poly(dG-dC).poly(dG-dC) as an indicator of Z-DNA binding.

Asunto(s)

RESUMEN

Three proteins from bull testis, previously thought to be Z-DNA-binding proteins but recently found to recognize brominated poly(dG-dC). poly(dG-dC) by criteria different from the Z-conformation, were partially sequenced. Of these, the 31 kDa protein was identified as a member of the high mobility group 2 protein family, and the 33 kDa protein as a member of the high mobility group 1 protein family. Both proteins had molecular weights approximately 30% higher than expected, indicating considerable posttranslational modification. In contrast, the 58 kDa protein remained unidentified for lack of any significant homology with known protein sequences.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Z-DNA has been detected in several pro- and eukaryotic cells and possible roles in regulating transcriptional activity and meiotic recombination proposed. The present study examined the localization of reaction product to potential Z-DNA sites in human testicular tubule epithelium from three subjects using an avidin-biotin complex (ABC)-immunoperoxidase method with a specific rabbit antibody previously shown to react with rat spermatogonial nuclei. A total of 46,626 cells were scored, of which 5656 were Sertoli cells. Eighty-six percent of spermatogonia in mitotic metaphase were found to be negative. Interphase spermatogonia identified as A dark or pale were positive in 93 and 92%, respectively, of cells, and this positivity persisted through B spermatogonia into meiosis. Of 8083 leptotene/zygotene spermatocytes, 99% were positive. Pachytene spermatocytes were 98% positive in the autosomal bivalents. First meiotic metaphase nuclei were negative (92%), as were almost all cells scored of the spermiogenic series (16,195). Nuclei of Sertoli cells had reaction product over the chromatin in 81% of 5656 cells, with no reaction product on the prominent nucleolus. The presence of potential Z-DNA sites in the genome of human spermatogenesis and Sertoli cells during known active stages of transcription (pachytene) and recombination (zygotene/pachytene) suggests a role for this conformation during these stages.

Asunto(s)

Asunto(s)

RESUMEN

Three Z-DNA-binding proteins of Mr 31, 33 and 58 kD were isolated from mature bull testis. They were obtained in a native state suitable for binding studies. These are the first examples of Z-DNA-binding proteins from a mammalian tissue. Purification involved tissue extraction with 0.35 M NaCl, cation exchange chromatography on CM-Trisacryl M and two consecutive anion exchange FPLC runs on Mono Q. The proteins appeared virtually homogeneous by anion exchange FPLC, SDS polyacrylamide gel electrophoresis and reverse phase HPLC (58 kD protein only). Yields from 50 g of testis tissue were: 31 kD protein, 40 micrograms; 33 kD protein, 100 micrograms; and 58 kD protein, 150 micrograms. Z-DNA binding was determined by Scatchard analysis of filter binding data using brominated poly(dG-dC).poly(dG-dC) as a conformation-specific ligand. Dissociation constants (Kz, in mol nucleotide/liter) were: 31 kD protein, 7 X 10(-7) M; 33 kD protein, 8 X 10(-7) M; 58 kD protein, 6 X 10(-8) M (primary binding site) and 6 X 10(-7) M (secondary binding site). B-DNA binding to poly(dG-dC).poly(dG-dG) was too low for reliable determination under the conditions of assay. This attested to a high degree of conformational specificity of the three proteins. The 58 kD protein bound Z-DNA at the primary site with an affinity almost equivalent to that of a polyclonal anti-Z-DNA antiserum raised in a rabbit (Kz, 4 X 10(-8) M).

Asunto(s)

RESUMEN

A single-stranded DNA-binding protein of Mr 35,000 (35K protein) was isolated from calf cerebral cortex by affinity chromatography on immobilized double-stranded and single-stranded DNA. Its localization in the nuclear compartment was demonstrated by immunohistochemistry. Previous studies had uncovered a homologous nonhistone chromosomal protein in the nuclei of rat cerebral cortex neurons, cerebellar neurons, oligodendrocytes, and liver cells. The rat protein accumulated in the nuclear compartment of neurons in exact temporal coincidence with the arrest of cell division and the initiation of terminal differentiation. Therefore, in the present work, the 35K protein was tested for an activating role in RNA transcription. During the course of this study we became aware that the 35K protein was identical to a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). When authentic GAPDH from rabbit skeletal muscle was injected into Xenopus laevis oocytes, it greatly stimulated RNA polymerase II transcription, whereas the 35K protein from calf brain did not. This apparent discrepancy was partially resolved by the finding that rabbit muscle GAPDH could be fractionated into two components by affinity chromatography on single-stranded DNA cellulose. Only 5% of the applied protein was retained on the column and could be eluted with a shallow salt gradient identical to the one used for the isolation of the 35K protein. This single-stranded DNA-binding component of rabbit muscle GAPDH did not stimulate transcription. Apparently, the 35K protein from calf brain corresponded to this single-stranded DNA-binding subfraction, which explained its failure to activate transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

A number of enzymes thought to be involved in DNA replication have been identified in the brain. These include single-stranded DNA-binding proteins, topoisomerases I and II, DNA polymerase alpha, a protein that binds Ap4A and might be classified as a DNA polymerase alpha accessory protein, RNase H, DNA polymerase beta, DNA ligase, an endo- and an exonuclease of unknown function, DNA methyl transferase and poly(ADPR) synthase. In contrast, little is known about the enzymology of DNA repair in brain. The few enzymes identified comprise uracil-DNA glycosylase, DNA polymerase beta, DNA polymerase alpha (which in neurons is present only at immature stages), DNA ligase, poly(ADPR) synthase, and O6-alkylguanine-DNA alkyltransferase. In addition, an exonuclease acting on depurinated single-stranded DNA (tentatively listed here as 3'----5' exonuclease), an endonuclease of unknown function as well as ill-defined acid and alkaline deoxyribonucleases also occur in brain.

Asunto(s)

RESUMEN

To test for metabolic deoxyribonucleic acid (DNA) turnover in differentiating neurons, [methyl-3H]thymidine was injected into the lateral cerebral ventricles of newly born rats, and after 6, 24 and 96 h, neuronal nuclei were prepared from the immature cerebral cortex. Enzymatic treatment converted virtually all of the DNA into soluble deoxynucleosides which were fractionated by high-performance liquid chromatography for determination of specific activity. The specific activity of thymidine was found to decline rapidly with time. The rate of this loss correlated with the radioactivity initially incorporated into the DNA. This suggested that DNA was being replaced by DNA repair as a consequence of radiation damage, rather than by spontaneous metabolic DNA turnover.

Asunto(s)

RESUMEN

Previous work from our laboratories has indicated that the DNA content of rat cerebral cortex neurons increases postnatally to a level of slightly above 3c, where 2c denotes the diploid DNA complement. We have re-evaluated this concept by using various cytophotometric assays and a novel high performance liquid chromatography (HPLC) technique. The latter consists of digesting the DNA in isolated neuronal nuclei by a mixture of DNA-degrading enzymes followed by analysis of the resulting deoxynucleosides by HPLC. We find that the various methods fall into two groups. The first gives evidence of a postnatal DNA (or histone) increase, while the second does not. The first group (DNA increase) comprises cytofluorometry for DNA following Schiff-type staining with fluorochromes 2,5-bis-(4-aminophenyl)-1,3,4-oxadiazole (BAO) and pararosaniline, ultraviolet absorption scanning for DNA and cytofluorometry for histones following staining with sulfaflavine at pH 8. The second group (no DNA increase) consists of cytofluorometry for DNA following staining with the DNA-complexing agents mithramycin, chromomycin A3, 4',6-diamidino-2-phenylindole (DAPI) and bisbenzimide (Hoechst 33258), as well as the newly developed HPLC technique. Since the HPLC technique measures DNA by a direct chemical approach without interference from other nuclear constituents or from higher order packaging in the chromatin, and detects at least 94-95% of the total DNA contained in neuronal nuclei independent of the developmental stage, we infer that the HPLC technique and, by consequence, the cytochemical assays of the second group reflect true DNA values. Therefore, we propose that cerebral cortex neurons retain a diploid DNA level throughout development.

Asunto(s)

Asunto(s)

RESUMEN

Recently, it has been shown that natural and synthetic deoxynucleotide polymers can adopt a left-handed helical structure (termed Z-DNA) in appropriate conditions (see, for example, refs 1 and 2 and the references therein). In contrast to the more familiar right-handed B-DNA, Z-DNA is strongly immunogenic, and polyclonal and monoclonal antibodies against Z-DNA have been elicited. By using such antibodies, immunoreactivity for Z-DNA has been detected in the polytene chromosomes of two dipteran species, in the macronucleus of a ciliated protozoon, and in certain plant nuclei (cited in ref. 11). In view of the possible importance of Z-DNA as a genomic regulatory signal, it would be highly desirable to know whether Z-DNA also occurs in mammals. We have therefore initiated an immunohistochemical study of various rat tissues by using three antisera specific for Z-DNA, and the peroxidase-antiperoxidase technique for visualization of tissue-bound antibodies. Here we demonstrate that the nuclei of many, but not all, types of rat cells exhibit Z-DNA immunoreactivity, suggesting that Z-DNA may exist naturally in mammalian chromatin.

Asunto(s)

Asunto(s)

RESUMEN

Single-stranded-DNA-binding proteins were analyzed in nuclei of differentiating rat cortex and cerebellar neurons. The developmental period investigated ranged from gestational day 19 (i.e. 3 days before term) to postnatal day 30. During this time both types of neurons undergo transition from proliferating, undifferentiated precursor cells to non-proliferating, terminally differentiated neurons. For comparison, nuclei from mature cortex glia and liver were also examined. Nuclei were isolated according to cell type, the proteins were 14C-labeled in vitro by reductive methylation and were fractionated by affinity chromatography on tandemly arranged columns of double-stranded and single-stranded DNA-cellulose. The columns were uncoupled and the proteins adsorbed to the single-stranded DNA were eluted with salt. They were then analyzed by high resolution two-dimensional gel electrophoresis followed by fluorography. This strategy ensured the selective detection of proteins that recognize single-stranded DNA specifically, and eliminated interference by proteins binding to DNA by simple ionic interaction as well as by proteins with affinity for double-stranded DNA. Many single-stranded-DNA-binding proteins showed conspicuous developmental fluctuations. In cortex neurons these took place around the time of birth and the first postnatal week, whereas in cerebellar neurons they occurred later and in a more protracted fashion. Thus, in both cortex and cerebellar neurons the protein changes followed a time course closely paralleling the arrest of cell division and the beginning of terminal differentiation. It is suggested that this approach may lead to the detection of putative regulatory proteins of the cell nucleus.

Asunto(s)

RESUMEN

Chromatin repeat lengths in neuronal, glial, and liver nuclei of the rat were determined by micrococcal nuclease digestion followed by gel electrophoresis. The repeat length of cortex neurons decreased from 200 base pairs (bp) before birth to 170 bp at 14 days and all subsequent stages. Administration of [3H]thymidine to pregnant rats during the period of fetal neurogenesis allowed neurons differing in their time of origin to be labeled individually. This revealed that the shortening of the chromatin repeat length affected only neurons generated early during development, i.e., between gestational days 13/14 and 18/19, whereas neurons continuing to proliferate beyond gestational day 19 and up to birth (day 22) did not undergo shortening of their repeat length. In contrast to the cortex neurons, cerebellar neurons (granule cells) underwent lengthening of the repeat length from 165 bp at fetal and early post-natal stages (up to day 4) to 218 bp after day 30. Thus, in both cortex and cerebellar neurons the changes occurred temporally coincident with major developmental processes. No changes were detected in liver nuclei during the same period. Non-astrocytic glia cells of the adult cortex had 200 bp repeats.

Asunto(s)

Asunto(s)