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Biomedical subjects

W E Hahn

Publications and source records attributed to W E Hahn.

At least 19 recordsLinked to original sources
PubMed1991

cDNA sequence and differential expression of the mouse Ca2+/calmodulin-dependent protein kinase IV gene.

We have isolated and sequenced a mouse brain cDNA encoding Ca2+/calmodulin-dependent protein kinase IV. The sequence predicts an acidic protein (pI = 4.56) of 469 amino acids (Mr = 52,627) that contains kinase catalytic and calmodulin-binding domains. The carboxy region has several primary structural features that suggest it may be a readily cleaved attachment domain. This region is highly charged and hydrophilic and contains several PEST sequences, motifs associated with high turnover proteins. Of the tissues examined, expression of the CaM kinase IV gene is restricted to brain and testis, where transcripts are differentially expressed to produce a kinase in both tissues and a calmodulin-binding protein, calspermin, in testis that lacks a kinase catalytic domain.

Amino Acid Sequence↗
PubMed1991

Identification of mRNAs associated with programmed cell death in immature thymocytes.

Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system. Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation. This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs. To search for possible death-associated mRNAs, we directionally cloned cDNA representing mRNA from control and dexamethasone-treated thymocytes. These libraries were used to produce ample amounts of DNA and RNA used in subtractive hybridization for the removal of sequences present in both control and induced cells. The remaining unhybridized sequences were selectively amplified by polymerase chain reaction and cloned to produce a library enriched for sequences expressed in death-induced cells. From this library we isolated cDNAs of death-associated mRNAs. One of these mRNAs, RP-8, appears within 1 h after exposure to gamma radiation, and a second mRNA, RP-2, is observed within 2 h. Both of these mRNAs accumulate during a period when a reference mRNA, actin, is declining. RP-2 and RP-8 are no longer detectable after 6 h postinduction, when apoptosis and mRNA degradation are evident in the culture. Sequence analysis of RP-8 cDNA indicates the presence of a zinc finger domain suggestive of a possible DNA regulatory role for the RP-8 protein. cDNA sequence results on RP-2 classify the corresponding protein as an integral membrane protein. We conclude that RP-2 and RP-8 are death-associated mRNAs that should be functionally evaluated in the context of the death process. As previously suggested, it may be that a family of "death genes" is activated by various stimuli depending on the type of cell, in a manner somewhat analogous to the induction of heat shock (stress) protein genes.

Amino Acid Sequence↗
PubMed1989

Sequence and expression of the murine diazepam binding inhibitor.

Previous studies suggest that a diazepam binding inhibitor (DBI, also referred to as endozepine) present in the brain may function anxiogenically as a modulator of the gamma-aminobutyric acid receptor complex (GABAA). An expression library representing mouse brain mRNA was screened using antisera that recognizes the 11 kDa DBI protein. A cDNA clone was isolated and sequenced. Comparison of the amino acid sequence of mouse DBI to that for human, rat and bovine DBI shows that the size of DBI is conserved at 87 amino acids in all of these mammals. DBI cDNA hybridizes to an mRNA of about 600 nucleotides. This mRNA is not restricted to the brain, being prevalent in other organs such as the liver and kidney. Its moderate to high abundance, as judged from mRNA levels, in several organs suggests that DBI might have functions other than, or in addition to, the possible regulation of benzodiazepine binding sites.

Amino Acid Sequence↗
PubMed1989

Chromosomal localization of the human gene for brain Ca2+/calmodulin-dependent protein kinase type IV.

Cloned cDNAs have been identified as corresponding to a new brain Ca2+/calmodulin-dependent protein kinase. On the basis of structural and immunological features, we refer to this new kinase as CaM Kinase IV. Two cDNA clones were used to identify CaM Kinase IV: The downstream clone, lambda ICM-1, contains the sequence encoding the calmodulin-binding site and the second clone, lambda ICM-2, encodes a partial amino acid sequence similar to the catalytic domain of several known protein kinases. Within the calmodulin-binding site a stretch of 8 amino acids (and 9 of 10) is identical to the corresponding site in the subunits of CaM Kinase II. Southern blot analysis shows the CaM Kinase IV gene is single copy in the mouse and human genomes. Synteny analysis of Southern blot data of DNA from hamster--human hybrid cells shows that the gene is present in human chromosome 5. Hybridization of cDNA probes to metaphase spreads of human chromosomes indicates that the gene is most likely located within the region of bands q21 to q23 of chromosome 5.

Adenomatous Polyposis Coli↗
PubMed1989

Isolation and sequence of a cDNA clone for human calcineurin B, the Ca2+-binding subunit of the Ca2+/calmodulin-stimulated protein phosphatase.

We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.

Amino Acid Sequence↗
PubMed1987

Screening an expression library with a ligand probe: isolation and sequence of a cDNA corresponding to a brain calmodulin-binding protein.

The use of cloning vectors that express inserted cDNA as fusion protein has led to the isolation of genes encoding a variety of eukaryotic proteins. In these instances antisera or monoclonal antibodies were used as probes to screen expression libraries. Since fusion proteins sometimes display biological activity reflective of the insert-specified portion, we tested the possibility that ligand-binding sites might exist in fusion proteins. Specifically we used 125I-labeled calmodulin as a probe to screen a mouse brain lambda gt11 library. One clone, lambda ICM-1 isolated using this approach, produces fusion protein that binds calmodulin with high affinity (Kd, 3-10 nM) in a Ca2+-dependent manner. Molecular genetic mapping experiments and deduction of the predicted higher-order structure from sequence data indicate the binding site is, or is within, a basic, amphiphilic alpha-helical domain composed of approximately 20 amino acids. lambda ICM-1 hybridizes with brain mRNA of 2.1 and 3.5 kb but not with mRNA from liver or kidney, suggesting possible restriction of the protein to brain. We discuss several observations that suggest lambda ICM-1 corresponds to Ca2+/calmodulin-dependent protein kinase II, an enzyme that phosphorylates several neuronal proteins, some of which apparently play a role in synaptic function. Our results suggest certain types of ligands may be useful probes to isolate genes encoding various receptor proteins, particularly when the protein is very rare or when it is difficult to obtain antibodies suitable for screening libraries.

Amino Acid Sequence↗
PubMed1985

Brain "identifier sequence" is not restricted to brain: similar abundance in nuclear RNA of other organs.

A repeated 82 base pair sequence in genomic DNA of the rat was previously proposed as being a control element governing brain (neuron) specific genetic expression. This intronic sequence, termed the brain "identifier" (ID), is complementary to small RNA species localized in brain cytoplasm, and it was thought to be represented specifically in RNA produced by brain nuclei in vitro. The RNA blot analyses of total nuclear and polyadenylated heterogeneous nuclear RNA described in the present report show that this ID sequence is also present in the liver and kidney in abundances similar to those in the brain. This repeated sequence is not, therefore, restricted to transcripts produced in the brain as suggested from previous transcriptional "runoff" experiments. Measurements on rat and mouse nuclear RNA indicate that the abundance of ID sequence transcript is roughly proportional to the number of copies of this repeat in the respective genomes. This suggests a rather random genomic location and transcription of this sequence. From these results it seems improbable that the ID sequence functions as a transcriptional-level control element in genes expressed specifically in the brain.

Animals↗
PubMed1983

Genetic expression in the developing brain.

The adult mouse brain contains complex populations of polyadenylated [poly(A)+] and nonpolyadenylated [poly(A)-] messenger RNA's (mRNA's). These mRNA's are separate sequence populations, similar in complexity, and in combination are equivalent to approximately 150,000 different mRNA sequences, of average length. Essentially all of the "adult" poly(A)+ mRNA's are present in the brain at birth. In contrast, most of the poly(A)- mRNA's are absent. Brain poly(A)- mRNA's begin to appear soon after birth, but the full adult complement is not reached until young adulthood. This suggests that these poly(A)- mRNA's specify proteins required for the biological capabilities of the brain that emerge during the course of postnatal development.

Animals↗
PubMed1982

Physical parameters affecting the rate and completion of RNA driven hybridization of DNA: new measurements relevant to quantitation based on kinetics.

Differences in the RNA-driven hybridization kinetics of genomic DNA and cDNA probes led us to examine physical parameters affecting these reactions. Cloned cDNA complementary to serum albumin (SA) mRNA hybridized in accordance with single component kinetics, whereas cloned SA genomic DNA hybridized more slowly and with multiple component kinetics. This difference is largely attributable to the relatively short and variable lengths of the mRNA complementary regions in the cloned genomic DNA. The rate of mRNA driven hybridization is affected to about half the extent observed for DNA renaturation as Na+ is increased or decreased from 0.18M. In the annealing of nucleic acids of high sequence complexity, after approximately 70% of reaction has been reached, the rate of the reaction is slowed and completion is not reached under "static" conditions. In practical terms, this is not the case for systems of low sequence complexity. This problem can be largely overcome by continuous or frequent mixing of the reactants, so that complex cDNA probes are hybridized essentially to completion, and kinetics can therefore be more readily compared to simple complexity standards.

Animals↗
PubMed1980

Sequence complexity of cDNA transcribed from a diverse mRNA population.

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.

Animals↗
PubMed1979

Complex population of nonpolyadenylated messenger RNA in mouse brain.

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.

Animals↗
PubMed1978

Complex population of mRNA sequences in large polyadenylylated nuclear RNA molecules.

Polyadenylylated heterogeneous nuclear RNA [poly(A)-hnRNA] from mouse brain was subjected to electrophoresis in agarose gels containing CH3HgOH, and molecules larger than 8 kilobases or 13 kilobases were recovered. cDNA was then transcribed from polyadenylylated RNA fragments cleaved from these large molecules. The resulting cDNA hybridized almost to completion with poly(A)-mRNA isolated from mouse brain polysomes. From the hybridization kinetics of this cDNA with its template RNA, it was estimated that the sequence complexity of the 3'-proximal sequences (of the same average size as mRNA) of the greater than 8 kilobase poly(A)-hnRNA was about 57,000 kilobases. The sequence complexity of poly(A)-mRNA, estimated from the template-driven hybridization kinetics of its respective cDNA, was about 110,000 kilobases. It is concluded that most, if not all, of the 3'-proximal sequences of large poly(A)-hnRNA molecules are homologous with mRNA in the mouse brain and that at least 40,000 different mRNA sequences (or portions of mRNA sequences) are represented in the 3'-proximal sequences of greater than 8 kilobase poly(A)-hnRNA.

Animals↗
PubMed1978

Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters.

A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters.

Animals↗
PubMed1977

One strand equivalent of the Escherichia coli genome is transcribed: complexity and abundance classes of mRNA.

DNA-RNA hybridization experiments show that essentially all of the genomic information is transcribed. High, intermediate, and rare abundance classes of messenger RNA (mRNA) are present, and their estimated complexities are equal to about 240, 1300, and 700 average-sized mRNA species, respectively. The high abundance mRNA species are present, an average, two to three copies per cell and constitute about 95 percent of the mRNA mass. Intermediate abundance mRNA species are present, on average, about once per 35 cells. The relative abundance and complexity of these mRNA classes correspond well with previous respective measurements on protein. Rare RNA species are thought to represent maximally repressed genes. Analysis of RNA synthesized in vitro by isolated nucleoids (chromosomes) suggests that sense and nonsense sequences are extensively interspersed on a given strand of the DNA.

Chromosome Mapping↗
PubMed1977

Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2' (3')-phosphate.

Severe degradation of high molecular weight RNA was shown to occur during incubation with commercially purified DNase. Most of the RNase activity could be removed by passage of the DNase through a column of agarose-coupled amino phenylphosphoryl-uridine-2' (3')-phosphate. Incubation with the treated DNase caused only minimal alteration of the sedimentation pattern of high molecular weight nuclear RNA, determined under partially denturing conditions. No impairment of DNase activity was detected.

Chromatography, Affinity↗