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J L Jorcano

Publications and source records attributed to J L Jorcano.

71 records · Page 4Linked to original sources
PubMed1984

Characterization of bovine keratin genes: similarities of exon patterns in genes coding for different keratins.

Four different genomic clones which contain the genes coding for epidermal keratins Ia (mol. wt. approximately 68 000), Ib (68 000), III (60 000) and VIb (54 500) have been selected using cDNA probes and identified by hybrid-selection translation. The genes vary considerably in length, primarily due to differences in intron sizes: keratin Ia, 9.3 kb (approximately 2.55 kb total exons); keratin Ib, 6.0 kb (2.25 kb exons); keratin III, 6.0 kb (2.2 kb exons); keratin VIb, 4.4 kb (1.85 kb exons). The genes for all three representatives of the basic (type II) cytokeratin subfamily, i.e., keratins Ia, Ib and III, contain eight introns of variable sizes (0.1-1.8 kb) and their exon patterns are very similar. The gene coding for keratin VIb, a representative of the acidic (type I) subfamily, contains seven introns, and the size pattern of its five innermost exons closely resembles that of the genes of the type II keratins. Most of the introns are located in regions coding for the alpha-helical cores of these proteins. Mapping of the intron positions by the S1 nuclease technique and sequencing of some exon-intron boundaries has revealed that some of the introns of all four keratin genes have similar positions to each other and to those of the hamster vimentin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I.

Cytoskeletal filaments of the alpha-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology. We compare two polypeptides of the acidic cytokeratin subfamily, VIb (Mr 54,000) and VII (Mr 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3' ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the alpha-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its alpha-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of Mr 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycine-rich and contain a canonic sequence GGGSGYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine Mr 59,000 keratin but is almost identical to the human cytokeratin number 14 of Mr 50,000, both in the alpha-helical and in the non-alpha-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3' non-coding ends. The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the alpha-helical and the non-helical regions as well as in the 3' non-coding portions of their mRNAs.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1984

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones.

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.

Animals↗
PubMed1984

Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences.

The flanking sequences of three U2 genes (or pseudogenes) and one U1 gene of Drosophila melanogaster have been determined. Comparison of the sequences reveals a remarkable homology between position -30 and -65 upstream from the structural genes, starting with a TATA box-like sequence. The 3' flanking regions are also conserved in all genes and contain a canonical A-A-T-A-A-A polyadenylation signal.

Animals↗
PubMed1984

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells.

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.

Animals↗
PubMed1984

Amino acid sequence diversity between bovine epidermal cytokeratin polypeptides of the basic (type II) subfamily as determined from cDNA clones.

The nucleotide sequences of four cDNA clones, each representing the carboxyterminal portion of a bovine epidermal cytokeratin of the "basic" (type II) subfamily, were determined, i.e., components Ia (Mr 68,000), Ib (Mr 68,000), III (Mr 60,000), and IV (Mr 59,000). The comparison of the sequences with each other and with the human type-II cytokeratin of Mr 56,000 reported by Hanukoglu and Fuchs [24] allows the following conclusions: The four major epidermal keratins of the basic (type II) subfamily, which are co-expressed in keratinocytes of the bovine muzzle, exhibit a high homology (greater than 90%) in the alpha-helical portion, but differ considerably in their nonhelical carboxy-terminal regions. The nonhelical carboxyterminal regions of all four cytokeratins are exceptionally rich in glycine and serine. Within the extrahelical tail, three different domains can be distinguished. The consensus sequence TYR(X)LLEGE which demarcates the end of the alpha-helical rod in all intermediate filaments is followed by a relatively short (22-27 amino acids) intercept rich in hydroxy amino acids and valine (carboxyterminal tail domain C1). This is followed by a long region that is variable in size and sequence, rich in glycine di-, tri-, and tetrapeptides, and contains diverse repeated sequences (domain C2). This is followed by another short (20 residues) hydroxy-amino-acid-rich intercept (domain C3) that ends with a conspicuously basic sequence of approximately four to six carboxyterminal amino acids. The first half of domain C1 is also homologous in all four keratins, suggesting that this region also assumes a common conformation and/or serves a special common function.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of Drosophila melanogaster U2 small nuclear RNA genes.

We describe here the organization of DNA sequences complementary to Drosophila melanogaster U2 small nuclear (sn) RNA. From a genomic library we isolated two recombinants containing two genes each. Genomic reconstruction experiments and Southern analysis revealed that D. melanogaster possesses only four to five U2 snRNA genes or very closely related sequences. The nucleotide sequence of one of the clones analysed shows 77% homology with rat U2 snRNA. A stretch of 12 nucleotides that has been implicated in heterogeneous nuclear RNA splicing is conserved between rat and Drosophila. The genomic organization of these genes is very similar in different melanogaster strains but diverges highly in different Drosophila species.

Animals↗
PubMed1983

Translational products of mRNAs coding for non-epidermal cytokeratins.

Total RNA and poly(A)+ RNA were isolated from tissues and cultured cells of various mammalian species (bovine muzzle epidermis and bladder urothelium; rat hepatoma cells; human cell lines HeLa, MCF-7 and A-431) and examined by translation in vitro using the reticulocyte lysate system. Polypeptides were separated and identified by two-dimensional electrophoresis and cytokeratins were selectively enriched from the translation assays by co-polymerization with added heterologous cytokeratins. In all three species, non-epidermal cytokeratins A, D and mol. wt. 40,000 (corresponding to numbers 8, 18 and 19 of the human cytokeratin catalog of Moll et al., 1982) were identified as translation products capable of co-polymerization with epidermal keratins. Several other basic and other acidic cytokeratins were also identified as translational products. In addition, two unidentified polypeptides (mol. wt. 52,000 and 43,000) which were minor polypeptides in cytoskeletons and translation assays were found to be specifically enriched in co-polymers with bovine epidermal keratins. The results indicate that many, perhaps all, non-epidermal cytokeratins characteristic of simple epithelia are genuine products of translation and that their diversity is not due to post-translational modification or processing. These findings, taken together with observations of in vitro translation of epidermal mRNAs, suggest that the diversity of cell type-specific expression of the different members of the cytokeratin polypeptide family is largely due to the cell type-specific synthesis of diverse mRNAs.

Animals↗
PubMed1983

Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.

Isolated desmosomes from bovine epidermis contain two major polypeptides of mol. wts. 75 000 (D6) and 83 000 (D5) which, like the desmoplakins of mol. wt. greater than 200 000, are associated with the insoluble desmosomal plaque structure. We have characterized these two polypeptides and examined their significance by peptide map comparisons and translation of bovine epidermal mRNA in vitro. Polypeptide D5 is different from polypeptide D6 by its apparent mol. wt., its isoelectric pH (approximately 6.35, whereas D6 is a basic polypeptide isoelectric at pH approximately 8.5) and its peptide map. By all these criteria desmosomal polypeptides D5 and D6 are also different from cytokeratins, desmoplakins and the glycosylated desmosomal proteins. Both polypeptides are synthesized from different mRNAs separable by gel electrophoresis on agarose: mRNA coding for polypeptide D5 is approximately 3500 nucleotides long, that for D6 is significantly shorter (estimated to 3050 nucleotides), and both contain relatively large proportions of non-coding sequences. The translational products of these mRNAs co-migrate, on two-dimensional gel electrophoresis, with the specific polypeptides from bovine epidermis, indicating that they are genuine polypeptides and are not the result of considerable post-translational processing or modification of precursor molecules. The cell and tissue distribution of these two cytoskeletal proteins and possible functions are discussed.

Animals↗
PubMed1983

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin.

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.

Animals↗
PubMed1980

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Chicken erythrocyte oligonucleosomes (trimers to about 20-mers) are able to interact with each other through the very lysine-rich histones (H1 and H5) and form heterogeneous globular particles with a mean diameter of about 300 A. These particles assemble spontaneously during micrococcal nuclease digestion of chromatin in the presence of 30 mM NaCl and contain approximately 25 nucleosomes. They are sensitive to ionic strength and unfold at lower salt concentrations but can be reconstituted by restoring the initial salt concentration. Even at 30 mM NaCl, the particles remain dynamic structures, being in equilibrium with their oligonucleosomal components as revealed by the fact that particle stability depends on the concentration of oligonucleosomes.

Animals↗
PubMed1979

An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.

The association of histones H2A, H2B, H3, and H4 in solution has been studied. In 2 M NaCl and at neutral pH they can assemble in a complex in which each histone is present in equimolar amounts. The complex has a weight average molecular weight of 98,000 (+/- 3700) and a sedimentation coefficient (so20,w) of 4.8. The value of the weight average molecular weight and the histone stoichiometry indicate that the complex is an octamer. The pairs of histones H2A,H2B and H3,H4 studied separately under identical conditions only associated as equimolar complexes consistent with dimeric and tetrameric structures, respectively. The stability of the core histone octamer is a function of the ionic strength, pH, and concentration of protein. The octamer dissociates by losing dimers of H2A,H2B until the main complexes existing in solution are the H3.H4 tetramer and the H2A.H2B dimer. This process is reversible upon reestablishing the original conditions.

Alkylation↗
PubMed1979

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.

Animals↗
PubMed1979

In vitro core particle and nucleosome assembly at physiological ionic strength.

Nucleosome core particles have been efficiently assembled in vitro by direct interaction of histones and DNA at physiological ionic strength, as assayed by digestion with DNases, supercoiling of relaxed circular DNA, and electron microscopy. Reconstitution was achieved either by the simultaneous addition of all core histones, or by the sequential binding of H3 . H4 tetramer and H2A . H2B dimer to DNA. Micrococcal nuclease digestion and electron microscopy studies indicated that there is heterogeneity in the spacings at which core particles are assembly on the DNA. Length measurements of oligomeric DNA produced during the course of the digestion suggest that the core histone octamer can organize 167 (+/- 4) rather than 145 base pairs of DNA, the extra 20 base pairs being quickly digested. Binding of histone H1 to core particles resulted in the protection of about 165 base pairs of DNA from nuclease attack. Because the core histone octamer is fully dissociated into H3 . H4 tetramer and H2A . H2B dimer at physiological ionic strength, our results would suggest that in vivo core particle assembly may also occur by interaction of these two complexes on the nascent DNA.

Animals↗
PubMed1978

Local perturbation of the double helical DNA structure near single breaks.

The influences of DNA composition and solvent modification on the stability of the native DNA structure near a single break have been studied. The value of the magnification parameter h of the double helix breakdown probability in a haplotomic degradation process is used as an index of the perturbation range. Methodological and technical details are discussed and comments are made on previously reported data on the h value. From this study it may be concluded that the width of the perturbation zone next to a nick is influenced by solvent-induced changes in the general stability of the native DNA molecule, and by compositional-dependent local interactions. Our results point to entropic effects as the more important contributions to stability perturbation near a discontinuity in the native DNA molecule.

DNA, Bacterial↗
PubMed1976

Hydrodynamic determination of polynucleotide chain discontinuities. Improved molecular weight correlations for denatured DNA.

Evidence is presented of the constancy in the conformation of denatured DNA in 2% formaldehyde in SSC (0.15 M NaCl/0.015 M trisodium citrate (pH 7.0)) over a wide range of molecular weights. It is also shown that denatured RNA behaves in the same way as DNA. The range of molecular weights studied runs from 0.02 to 16 X 10(6). In accordance with these results, biparametric expressions are proposed for molecular weight calculations from sedimentation or viscosity data of denatured DNA or RNA, when determined in 2% formaldehyde in SSC. Testing of the expressions with standard DNA and RNA preparations showed good correlation.

Bacillus subtilis↗