Biomedical subjects

D F Clayton

Publications and source records attributed to D F Clayton.

At least 37 records · Page 2Linked to original sources

PubMed1995

Differential induction of the ZENK gene in the avian forebrain and song control circuit after metrazole-induced depolarization.

ZENK is an immediate early gene (IEG) that encodes a transcription factor protein, and its induction has been proposed as a necessary step in the cellular process underlying long-term memory formation. We have previously shown that ZENK is induced in adult songbird brain by the sound of birdsong, but, interestingly, induction did not occur in several areas known to respond to song stimuli. Conceivably, the ZENK gene may be repressed in these areas in adult birds. As a further test of this hypothesis, we administered metrazole, a strong GABAergic antagonist that leads to widespread excitation in the brain. Following metrazole, ZENK mRNA increases more than 10-fold throughout most of the telencephalon in both canaries and zebra finches, and primarily in neurons. In contrast, ZENK induction is much lower or absent in the archistriatum, the primary telencephalic sensory-recipient areas (including auditory field L), and the three telencephalic androgen receptor-containing song nuclei (high vocal center, lateral magnocellular nucleus of the anterior neostriatum, and the robust nucleus of the archistriatum). We did not observe any differences in ZENK induction patterns in juvenile versus adult zebra finches, or fall versus spring male canaries. Together with our previous studies of induction by song, these results suggest that in specific parts of the forebrain, including most of the song control system, IEG expression is subject to different constraints than in the rest of the forebrain. Understanding the molecular basis for this differential gene regulation may prove invaluable in understanding the organization of the song control circuit and the avian telencephalon.

Animals↗

PubMed1995

The canary androgen receptor mRNA is localized in the song control nuclei of the brain and is rapidly regulated by testosterone.

Singing in canaries is an androgen-inducible behavior, under the control of an identified motor pathway, which includes several discrete "song nuclei" in the telencephalon. To determine whether the mRNA for the canary androgen receptor (cAR) is expressed in these song control nuclei, we synthesized probes from the recently cloned cAR cDNA and used in situ hybridization to examine spring male canary brain sections. Concentrations of cAR mRNA are detectable in several of the song control nuclei of the forebrain, including high vocal center (HVC), lateral magnocellular nucleus of the anterior neostriatum and robust nucleus of the archistriatum. In addition, we also show that testosterone treatment rapidly induces a significant reduction of cAR mRNA levels in nucleus HVC of females. Since the effects of androgen on singing behavior occur much more slowly, the behavioral effects are probably a secondary or independent result of androgen's primary and immediate action on target gene transcription.

Animals↗

PubMed1995

Correspondence between sites of NGFI-A induction and sites of morphological plasticity following exposure to environmental complexity.

To determine if gene regulation may play a role in behaviorally-induced morphological plasticity in the brain, we used in situ hybridization to measure levels of mRNA for the immediate early gene transcription factor NGFI-A (also known as ZENK, zif/268, egr-1 and Krox 24). Brains of periadolescent male rats exposed to 2-4 days of the following behavioral treatments were compared: (1) group housing in a complex environment (EC); (2) individual housing with daily handling (HIC); and (3) individual handling (IC). Quantitative analysis of the autoradiograms revealed that EC rats had significantly higher levels of NGFI-A than IC rats in regions of cortex previously shown to exhibit morphological plasticity (most pronounced in visual cortex), but not in frontal cortex where no dendritic changes have been detected. HIC rats were intermediate between the two groups. These data support an association between structural plasticity and altered patterns of immediate early gene expression.

Animals↗

PubMed1995

Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.

A male zebra finch learns a song by listening to a tutor, but song learning is normally restricted to a critical period in juvenile development. Here we identify an RNA whose expression in the song control circuit is altered during this critical period. The RNA encodes a soluble presynaptic protein that forms a predicted amphipathic alpha helix typical of the lipid-binding domain in apolipoproteins. We show this protein, which we call synelfin, to be the homolog of the human non-A beta component (and its precursor) recently purified from Alzheimer's disease amyloid. We suggest this highly conserved protein may serve a novel function critical to the regulation of vertebrate neural plasticity.

Alzheimer Disease↗

PubMed1994

Immediate-early gene responses in the avian song control system: cloning and expression analysis of the canary c-jun cDNA.

Previous studies have shown that song presentation results in a rapid rise in mRNA levels for the ZENK gene (the avian homologue of zif-268, Egr-1, NGFI-A, and Krox-24) in specific parts of the songbird forbrain. Metrazole-induced seizures also cause an increase in ZENK mRNA, even more widely throughout the telencephalon. Surprisingly, however, little or no ZENK induction by either stimulus was observed in several forebrain areas involved in auditory processing and song production. To learn whether this pattern of regulation is specific to ZENK, we examined the response of another 'immediate-early' gene, c-jun. Here we first describe the identification, cloning and sequence analysis of a canary cDNA encoding c-jun. Then, by in situ hybridization we show that c-jun is also induced by song or seizure, and in a pattern mostly similar to ZENK. As with ZENK, no induction of c-jun is observed in the androgen receptor-containing song nuclei or within the primary thalamo-recipient auditory area of the forebrain. Thus common immediate early gene responses appear to be selectively uncoupled from physiological activation in these specific forebrain regions, which are also characterized by tight developmental, hormonal and seasonal regulation.

Animals↗

PubMed1994

Seasonal and tissue-specific regulation of canary androgen receptor messenger ribonucleic acid.

Natural seasonal fluctuations in androgen levels appear to cause changes in physiology and reproductive behavior, such as singing, in canaries. Little is known, however, about the cellular mechanisms underlying these changes. Because androgens act principally through nuclear receptors in other species, we have isolated and sequenced a cDNA likely to encode the canary androgen receptor and used this cDNA to examine the regulation of AR mRNA levels in the testis, kidney, and liver of the canary. The sequence corresponds to most of the coding portion of seven of the eight exons found in the homologous mammalian gene, including the domains that bind to DNA and androgen and affect transcription. Its mRNA is approximately 8 kilobases in length and is encoded by a single gene. In the testis, the transcript is expressed specifically in the Sertoli cells. The androgen receptor antagonist flutamide represses AR mRNA levels in kidney, but induces them in liver, indicating that androgen regulates its receptor, but does so in a tissue-specific manner, as is seen for the estrogen receptor in rodents. In addition, there are natural seasonal fluctuations in AR mRNA levels in testis and liver correlated with seasonal differences in the levels of circulating androgens. This is the first evidence of natural feedback regulation of AR mRNA levels.

Amino Acid Sequence↗

PubMed1994

Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system.

The ZENK gene encodes a zinc-finger-containing transcriptional regulator and can be rapidly activated in songbird brain by presentation of birdsong (Mello et al., 1992). Here we map the areas of the songbird forebrain that show this genomic response to birdsong, using in situ hybridization. After 30 min of song presentation ZENK mRNA levels reach a peak in the caudomedial telencephalon, in areas adjacent to or closely related with primary auditory structures. These areas include subfields of field L (L1 and L3), the caudomedial neostriatum (NCM), the caudomedial hyperstriatum ventrale (CMHV) anterior to field L, the caudal paleostriatum, and two field L targets, HVC shelf and RA cup. In contrast, ZENK induction is absent in some areas that show a response to song by other measures and where ZENK induction might have been expected. These include the direct thalamo-recipient field L subfield L2, and the nuclei of the circuit involved in the acquisition and production of learned song. These results demonstrate that ZENK induction following song presentation occurs only in a subset of areas physiologically activated by song, and draw attention to areas previously unsuspected as related to processing of complex auditory stimuli. Based on what is known about ZENK function in mammalian systems (Christy et al., 1989; Cole et al., 1989; Wisden et al., 1990), we speculate that areas revealed by ZENK induction might correspond to sites where critical neuronal modifications occur in response to birdsong presentation, possibly leading to the formation of song-related memories.

Animals↗

PubMed1992

Song presentation induces gene expression in the songbird forebrain.

We investigated the participation of genomic regulatory events in the response of the songbird brain to a natural auditory stimulus of known physiological and behavioral relevance, birdsong. Using in situ hybridization, we detected a rapid increase in forebrain mRNA levels of an immediate-early gene encoding a transcriptional regulator (ZENK; also known as zif-268, egr-1, NGFI-A, or Krox-24) following presentation of tape-recorded songs to canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). ZENK induction is most marked in a forebrain region believed to participate in auditory processing and is greatest when birds hear the song of their own species. A significantly lower level of induction occurs when birds hear the song of a different species and no induction is seen after exposure to tone bursts. Cellular analysis indicates that the level of induction reflects the proportion of neurons recruited to express the gene. These results suggest a role for genomic responses in neural processes linked to song pattern recognition, discrimination, or the formation of auditory associations.

Acoustic Stimulation↗

PubMed1992

Differential regulation in the avian song control circuit of an mRNA predicting a highly conserved protein related to protein kinase C and the bcr oncogene.

An RNA identified by differential cDNA cloning (HAT-2) is highly enriched in canary forebrain in areas associated with the control of complex learned behaviors and higher perceptual processes. The nucleotide sequence predicts a protein that is 96% identical to the product of the n-chimaerin gene isolated from human brain and contains two identifiable domains suggesting a novel role in signal transduction processes. One domain is similar to the sequence in protein kinase C which mediates diacylglycerol binding and regulation. The second domain is similar to a portion of BCR, a GTPase-activating protein encoded by the breakpoint cluster region gene. In male canaries examined during the song season, HAT-2 RNA shows variable expression within the song control circuit, and is notably less abundant in the three nuclei which concentrate androgens (HVC, RA and L-MAN). A fundamental function in the vertebrate forebrain and a possible role in the regulation of neural plasticity are suggested by the conserved structure and pattern of expression of this gene in the brain.

Amino Acid Sequence↗

PubMed1991

Structure and expression of canary myc family genes.

We found that the canary N-myc gene is highly related to mammalian N-myc genes in both the protein-coding region and the long 3' untranslated region. Examined coding regions of the canary c-myc gene were also highly related to their mammalian counterparts, but in contrast to N-myc, the canary and mammalian c-myc genes were quite divergent in their 3' untranslated regions. We readily detected N-myc and c-myc expression in the adult canary brain and found N-myc expression both at sites of proliferating neuronal precursors and in mature neurons.

Amino Acid Sequence↗

PubMed1990

Forebrain-enriched RNAs of the canary: a population analysis using hybridization kinetics.

Canaries and other songbirds offer unique advantages for analyzing the relationship between specific gene regulation and neural plasticity. To establish a quantitative profile of the population of RNAs potentially involved in this regulation, we analyzed the solution hybridization kinetics of canary forebrain cytoplasmic polyadenylated RNA. Hybridization of forebrain cDNA to forebrain RNA provides evidence for RNA species at individual concentrations ranging from less than 10(-6) to about 10(-3) (by fractional mass). Cross-hybridization to RNA from the rest of the brain, together with other studies, defines a subpopulation of about 5000 rare RNAs that are enriched in the forebrain compared to the rest of the brain. Some of these forebrain-enriched RNAs are likely to play a role in regulating the neural plasticity characteristic of the canary forebrain.

Animals↗

PubMed1989

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity.

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.

Animals↗

PubMed1988

Probes for rare mRNAs reveal distributed cell subsets in canary brain.

cDNA clones of 7 low-abundance canary brain RNAs hybridize in situ to different subsets of brain cells. Although these cell sets are distinct, they are dispersed in a variety of brain regions with overlapping anatomical distributions. These cDNA clones were initially selected by their relative hybridization to forebrain and rest-of-brain RNAs and represent a sampling of a much larger population of differentially expressed RNAs present at individual concentrations of 10(-7) to 10(-4) as a fraction of polyadenylated RNA mass. Our results suggest the existence of several thousand low-abundance brain mRNAs likely to be distributed in diverse and overlapping brain cell subsets. Furthermore, our experiments define a simple and general strategy for producing and analyzing molecular probes for subsets of brain cells and provide an initial set of useful reagents for further study of brain organization and development.

Animals↗

PubMed1987

Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences.

The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.

Acetylation↗

PubMed1986

Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression.

Recombinant adenoviruses were constructed in which the viral E1A gene was deleted and the E1B promoter was replaced by the rat albumin, mouse beta-major globin, or mouse immunoglobulin heavy-chain promoter. After infection of human or rat hepatoma cells, E1B-containing mRNAs could be detected only from the virus containing the albumin promoter. Conversely, only the immunoglobulin promoter was active in virus-infected myeloma cells. However, in hepatoma cells transcription from the albumin promoter in the virus was much less than that of the endogenous cellular albumin gene or of other viral genes. In primary mouse hepatocytes endogenous albumin gene transcription was high immediately after plating but declined within 24 h. Expression of the albumin promoter in the virus paralleled that of the cellular albumin gene. From these results it appears that cell-specific expression of albumin depends on the presence of tissue-specific trans-acting factors, but the presence of such factors does not suffice for a maximal rate of transcription, a conclusion that requires direct comparison within a differentiated cell of a newly introduced and preexisting active cell gene.

Adenovirus Early Proteins↗

PubMed1985

Dependence of liver-specific transcription on tissue organization.

When the liver is disaggregated and hepatocytes are cultured as a cellular monolayer for 24 h, a sharp decline (80 to 99% decrease) in the transcription of most liver-specific mRNAs, but not common mRNAs, occurs (Clayton and Darnell, Mol. Cell. Biol. 2:1552-1561, 1983). A wide variety of culture conditions involving various hormones and substrates and cocultivation with other cells failed to sustain high rates of liver-specific mRNA synthesis in cultured hepatocytes, although they continued to synthesize common mRNAs at normal or elevated rates. In contrast, when slices of intact mouse liver tissue were placed in culture, the transcription of liver-specific genes was maintained at high levels (20 to 100% of normal liver). Furthermore, we found that cells in the liver could be disengaged and immediately reengaged in a tissue-like structure by perfusing the liver with EDTA followed by serum-containing culture medium. Slices of reengaged liver continued to transcribe tissue-specific mRNA sequences at significantly higher rates after 24 h in culture than did individual cells isolated by EDTA perfusion followed by culturing as a monolayer. Therefore we conclude that a mature tissue structure plays an important role in the maintenance of maximum tissue-specific transcription in liver cells.

Animals↗

PubMed1985

Liver-specific RNA metabolism in hepatoma cells: variations in transcription rates and mRNA levels.

The transcription rate and abundance of several liver-specific mRNAs as well as mRNAs common to many cell types were compared in a series of rodent hepatoma cell lines, normal liver cells, and primary hepatocyte cultures. The rat hepatoma cell line, Fao, which displays a liver-specific phenotype, contained eight of eight liver-specific mRNAs examined. However, the transcription rates of most liver-specific mRNAs were found to be low (1 to 30%) compared with normal liver in this and other differentiated cell lines. This low rate is similar to the transcription rates of liver-specific mRNA sequences measured in primary cultures of hepatocytes. Several variant cell lines that had lost differentiated traits contained few or none of the liver-specific mRNAs; clonal descendents which had regained differentiated function regained the tissue-specific mRNAs as a group, but at various concentrations. Because all of the changes observed in mRNA levels were not accompanied by parallel changes in transcription of the same sequences, differential posttranscriptional stabilization of the liver-specific mRNAs must also occur in the different cell lines. These results qualify the utility of cultured cell lines in the study of tissue-specific transcriptional control, but raise the possibility that posttranscriptional mechanisms act in cooperation with transcriptional controls to bring the level of tissue-specific mRNAs closer to those found in liver cells.

Albumins↗

PubMed1984

Posttranscriptional modulation of gene expression in cultured rat hepatocytes.

The maintenance of high levels of two liver-specific mRNAs in cultured hepatocytes was achieved in a serum-free hormonally defined cell culture medium. However, this maintenance of liver-specific mRNA levels did not correlate with the level of transcription of the genes but was apparently due to increased stabilization of the tissue-specific mRNAs. The mRNA stabilization did not occur in serum-supplemented medium. In both defined and serum-supplemented medium, actin and tubulin mRNAs were also greatly increased, in both cases predominantly if not entirely due to increased mRNA stability.

Actins↗