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M Blumenberg

Publications and source records attributed to M Blumenberg.

52 records · Page 3Linked to original sources
PubMed1990

Epithelial-specific keratin gene expression: identification of a 300 base-pair controlling segment.

To elucidate the elements required for regulation of keratin expression in epidermis, we have linked a short, 300 base pair segment, corresponding to the promoter region of a human K#14 gene, to the chloramphenicol-acetyl-transferase gene. This construct was introduced into various mammalian cell lines and primary cultures via Ca3(PO4)2 precipitation. The 300 base pair segment from the keratin gene promoter region was active in all epithelial cells studied including transformed, simple epithelial cells such as HeLa and ME-180, cell lines derived from stratified epithelium, such as SCC-12, as well as primary cultures of epithelial cells. The construct was inactive in all non-epithelial cells tested including fibroblasts and melanocytes. The segment does not function as a silencer in nonepithelial cells but it can function as an enhancer in epithelial cells. Using the polymerase chain reaction we have constructed a series of deletions of the promoter and have localized an essential function within a 40 bp sequence. We conclude that we have identified the keratin gene promoter that is sufficient to confer epithelial-specific expression.

Animals↗
PubMed1990

Three parallel linkage groups of human acidic keratin genes.

Two regions of human genomic DNA, each containing several keratin genes, were isolated and partially sequenced. The keratin genes are inactive, having suffered deleterious mutations. Both regions contain at least four keratin genes arranged in a head-to-tail orientation including a pseudogene for keratin K#16. Within each segment there are two keratin genes in close linkage with only 1.5 kb of DNA between them. Sequence comparison of the two regions showed 98.9% identity in both the coding and the intronic segments of the pseudogenes. The pseudogenes show 94% identity to their functional counterparts. Southern hybridization analysis showed that the segments are paralogous, not allelic. The regions are products of two independent, recent duplication events. The first occurred approximately 24 million years ago, after the separation of primates from the rhesus/baboon line. The second is specific for the human lineage, having occurred approximately 3.8 million years ago. Analysis of the genomic DNAs of primates showed the presence of only one of the regions in the DNAs of gibbon and gorilla, while rhesus monkey and baboon were missing both copies. We conclude that the human keratin genes are still actively evolving, with new duplications having occurred as recently as after the separation of human and gorilla ancestors.

Amino Acid Sequence↗
PubMed1990

Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes.

In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters.

Animals↗
PubMed1989

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins.

The earliest gene duplications in the evolution of the intermediate filament proteins created the ancestors of acidic keratins, basic keratins, nonepithelial intermediate filament proteins, and lamins. Biochemistry and function of cytoplasmic intermediate filaments differ greatly from those of lamins. Cytoplasmic intermediate filament proteins have a different cellular location than lamins, form different types of supramolecular structures, and are missing a protein segment found in lamins; but the data presented here indicate that the cytoplasmic intermediate filaments do not have a common ancestor separate from the ancestor of lamins. In the non-epithelial intermediate filament branch, the ancestor of neurofilament proteins and the common ancestor of desmin, vimentin, and glial fibrillary acidic protein (GFAP) diverged first. By evolutionary criteria, the intermediate filament protein recently discovered in neuronal cells does not belong to the neurofilament family but is more closely related to desmin, vimentin, and GFAP. Sequences of different sub-domains yield different evolutionary trees, possibly indicating existence of sub-domain-specific functions.

Amino Acid Sequence↗
PubMed1988

Concerted gene duplications in the two keratin gene families.

Evolutionary trees were derived from the keratin protein sequences using the Phylogeny Analysis Using Parsimony (PAUP) set of programs. Three major unexpected conclusions were derived from the analysis: The smallest keratin protein subunit, K#19 (Moll et al. 1982), is not the most primitive one, but has evolved to fulfill a highly specialized function, presumably to redress the unbalanced synthesis of keratin subunits. Second, the ancestors of keratins expressed in the early embryonic stages, K#8 and K#18, were the first to diverge from the ancestors of all the other keratins. The branches leading to these two keratins are relatively short, indicating a comparatively strong selection against changes in the sequences of these two proteins. Third, the two keratin families show extraordinary parallelism in their patterns of gene duplications. In both families the genes expressed in embryos diverged first, later bursts of gene duplications created the subfamilies expressed in various differentiated cells, and relatively recent gene duplications gave rise to the hair keratin genes and separated the basal cell-specific keratin from those expressed under hyperproliferative conditions. The parallelism of gene duplications in the two keratin gene families implies a mechanism in which duplications in one family influence duplication events in the other family.

Animals↗
PubMed1988

Inactivation of human keratin genes: the spectrum of mutations in the sequence of an acidic keratin pseudogene.

Keratins are cytoskeletal proteins encoded by a multigene family. We have identified the first human keratin pseudogene and determined its complete nucleotide sequence. Sequence comparisons indicate that the pseudogene arose from a very recent duplication of the 50-kd keratin (K14) gene. The coding and the intron sequences of the two genes are 95% and 93% identical, respectively. Although the sequence of the regulatory region in the pseudogene is virtually identical to that in the 50-kd functional gene, several deleterious mutations have been identified in the pseudogene. There are three frameshifts in the coding regions, one of which is a perfect 8-bp duplication. A single-base-pair deletion in the first exon and a single-base-pair insertion in the penultimate exon also result in frameshifts. The three remaining deleterious mutations interfere with the mRNA processing signals: two alter the intron/exon boundaries, and the third disrupts the polyadenylation signal. These mutations clearly identify the sequence as a human keratin pseudogene.

Base Sequence↗
PubMed1988

Embryonic expression of the human 40-kD keratin: evidence from a processed pseudogene sequence.

Analysis of the cytoskeletal components of early murine embryos has detected expression of two keratin proteins, K#8 and K#18, at the 4-8-cell stage. Comparable data for human embryos do not exist, although several processed pseudogenes corresponding to K#8 and K#18 have been discovered in the human genome. Because only genes that are expressed in pre-germ-line and germ-line cells can give rise to processed pseudogenes, the existence of human K#8 and K#18 processed pseudogenes is prima facie evidence for expression of keratins K#8 and K#18 in the early human embryo. We have cloned and determined the complete sequence of a processed pseudogene corresponding to another acidic human keratin. Comparison of its sequence with known sequences of other mammalian keratins indicates that the pseudogene arose from a reverse transcript of a correctly initiated and terminated functional human K#19 gene. This implies expression of K#19 keratin in addition to K#8 and K#18 in the early human embryo. We have proposed previously that K#19 evolved specifically to redress unbalanced production of various basic keratins, and our current evidence, that it is expressed at an early stage of development, implies that K#19 may fulfill this same role during human embryogenesis.

Amino Acid Sequence↗
PubMed1987

Evolution of keratin genes: different protein domains evolve by different pathways.

Intermediate filaments are composed of a family of proteins that evolved from a common ancestor. The proteins consist of three domains: a central, alpha-helical domain similar in all intermediate filaments, bracketed by two domains that are variable in length and structure. Within the intermediate-filament family, several subfamilies have been recognized by immunologic and nucleic acid hybridization techniques. In this paper we present the sequence of the genomic DNA coding for a 65-kilodalton human keratin and compare it with the sequences of other intermediate-filament proteins. While the central, alpha-helical domains of these proteins show homologies that indicate a common ancestor, the sequences of the variable terminal domains indicate that the variable domains evolved through a series of tandem duplications and possibly by gene-conversion mechanisms.

Amino Acid Sequence↗
PubMed1986

Linkage of human keratin genes.

Two families of keratins, type I and type II, can be distinguished within the intermediate filament family of proteins, and at least 20 genes in the human genome code for the 20 known keratin proteins. In epithelial intermediate filaments, keratins from both families appear to be coordinately expressed. We have screened a library of human genomic DNA and have identified several cases of linkage among homologous and heterologous pairs of keratin genes. Genes coding for type I keratins were found linked to those coding for type II keratins. Linkage was discovered also among homologous genes coding for type I keratins and among genes encoding type II keratins. In addition, we found genes coding for glycine-rich keratins linked to genes coding for those that do not contain glycine-rich regions. Our results raise the possibility that all keratin genes are linked in a single region of the human genome.

Bacteriophage lambda↗
PubMed1985

A systematic approach for detecting high-frequency restriction fragment length polymorphisms using large genomic probes.

Thirteen phage clones containing low-copy sequences were isolated from a human DNA library and tested for their ability to detect restriction fragment length polymorphisms (RFLPs). Reported are the RFLPs revealed with each clone, all found in frequencies useful for linkage studies. Cytological data are available for five of the 13 clones, with regional assignments made for three of the markers by in situ hybridization. It is concluded that phage clones containing large unique DNA inserts detect multiple RFLPs with high efficiency. An analysis of the relative efficiency of 20 restriction enzymes for detecting single nucleotide changes is discussed by comparing the observed data to those expected on the basis of recognition and potential site frequencies, as computed from the dinucleotide distribution. Finally, in an effort to facilitate linkage studies using polymorphic DNA sequences, experiments were made with pools of probes from various sources.

Bacteriophages↗
PubMed1982

Regulatory region of the Klebsiella aerogenes tryptophan operon.

The trp operon of Klebsiella aerogenes was cloned, and its regulatory region was sequenced. Comparison with previously reported trp regulatory sequences of other enteric bacteria indicates that the K. aerogenes trp promoter-operator region is most similar to the corresponding region of Salmonella typhimurium. The trp leader regions of K. aerogenes and other enteric bacteria are organized similarly, but there are significant differences in the stabilities of the predicted secondary structures in their leader transcripts. These differences should make the K. aerogenes attenuator a weaker transcription termination site than any of the other attenuator regions studied; this was confirmed in in vitro transcription experiments. The sequence of the leader transcript and the precise site of in vitro termination were determined.

Base Sequence↗
PubMed1982

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria.

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.

Anthranilate Synthase↗
PubMed1981

Comparison of the nucleoside sequence of trpA and sequences immediately beyond the trp operon of Klebsiella aerogenes. Salmonella typhimurium and Escherichia coli.

The nucleotide sequence of trpA of Klebsiella aerogenes is presented and compared with the trpA sequences of Salmonella typhimurium and Escherichia coli. The majority of the approximately 200 differences between each pair of trpA's are single nucleotide pair changes that do not alter the amino acid sequence. Codon usage conforms to the general patterns revealed by examination of other prokaryotic gene sequences. However, codon usage in K. aerogenes trpA reflects the high G+C content of the genome of this organism. The DNA sequences just beyond trpA, the presumed transcription termination region, are also compared for the three species. Perusal of these sequences indicates that the secondary structure of the transcript segment just beyond trpA has been preserved, while the primary sequence has diverged appreciably.

Amino Acid Sequence↗
PubMed1981

Physical maps of Klebsiella aerogenes and Salmonella typhimurium hut genes.

The recognition sites for several restriction endonucleases were mapped within deoxyribonucleic acid coding for histidine utilization (hut) genes of Salmonella typhimurium and Klebsiella aerogenes. Deoxyribonucleic acid fragments containing the two hut promoters were identified by ribonucleic acid polymerase binding.

DNA Restriction Enzymes↗
PubMed1977

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.

Adenosine Monophosphate↗