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R C Scarpulla

Publications and source records attributed to R C Scarpulla.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nuclear respiratory factor 1 activation sites in genes encoding the gamma-subunit of ATP synthase, eukaryotic initiation factor 2 alpha, and tyrosine aminotransferase. Specific interaction of purified NRF-1 with multiple target genes.

Transcription factor nuclear respiratory factor 1 (NRF-1) was originally identified as an activator of the cytochrome c gene and subsequently found to stimulate transcription through specific sites in other nuclear genes whose products function in the mitochondria. These include subunits of the cytochrome oxidase and reductase complexes and a component of the mitochondrial DNA replication machinery. Here we establish that a functional recognition site for NRF-1 is present in the ATP synthase gamma-subunit gene extending the proposed respiratory role of NRF-1 to complex V. In addition, biologically active NRF-1 sites are found in genes encoding the eukaryotic translation initiation factor 2 alpha-subunit and tyrosine aminotransferase, both of which participate in the rate-limiting step of their respective pathways of protein biosynthesis and tyrosine catabolism. The recognition sites from each of these genes form identical complexes with NRF-1 as established by competition binding assays, methylation interference footprinting, and UV-induced DNA cross-linking. Cloned oligomers of each NRF-1 binding site also stimulate the activity of a truncated cytochrome c promoter in transfected cells. The NRF-1 binding activities for the various target sites copurified approximately 33,000-fold and resided in a single protein of 68 kDa. These observations further support a role for NRF-1 in the expression of nuclear respiratory genes and suggest it may help coordinate respiratory metabolism with other biosynthetic and degradative pathways.

Animals↗
PubMed1991

Transcriptional activation through ETS domain binding sites in the cytochrome c oxidase subunit IV gene.

A mutational analysis of the rat cytochrome c oxidase subunit IV (RCO4) promoter region revealed the presence of a major control element consisting of a tandemly repeated pair of binding sites for a nuclear factor from HeLa cells. This factor was designated NRF-2 (nuclear respiratory factor 2) because a functional recognition site was also found in the human ATP synthase beta-subunit gene. Deletion or site-directed point mutations of the NRF-2 binding sites in the RCO4 promoter resulted in substantial loss of transcriptional activity, and synthetic oligomers of the NRF-2 binding sites from both genes stimulated a heterologous promoter when cloned in cis. NRF-2 binding and transcriptional activation required a purine-rich core sequence, GGAA. This motif is characteristic of the recognition site for a family of activators referred to as ETS domain proteins because of the similarity within their DNA-binding domains to the ets-1 proto-oncogene product. NRF-2 recognized an authentic Ets-1 site within the Moloney murine sarcoma virus long terminal repeat, and this site was able to compete for NRF-2 binding to the RCO4 promoter sequence. In addition, a single polypeptide of 55 kDa was detected following cross-linking of a partially purified NRF-2 fraction to RCO4, the human ATP synthase beta subunit, or Moloney murine sarcoma virus binding sites. However, in contrast to Ets-1, which appears to be exclusive to lymphoid tissues, NRF-2 has the broad tissue distribution expected of a regulator of respiratory chain expression.

Animals↗
PubMed1990

The rat cytochrome c oxidase subunit IV gene family: tissue-specific and hormonal differences in subunit IV and cytochrome c mRNA expression.

We have isolated three members of the rat cytochrome c oxidase subunit IV gene family: one functional gene and two processed pseudogenes. The pseudogenes appear to represent the only other closely related sequences in this family. The functional gene encodes an isoform which is expressed in all tissues examined and has features characteristic of 'housekeeping' genes. These include multiple transcription start sites mapped to within an approximately 50 bp region and a GC-rich promoter lacking typical CCAAT or TATAA sequences. Although the subunit IV gene is expressed at its highest levels in cardiac and skeletal muscle, consistent with the high energy demand in those tissues, its expression differs from that of cytochrome c in several respects. 1) Subunit IV mRNA abundance in various tissues is relatively uniform when compared to the highly variable levels of cytochrome c mRNAs. 2) Unlike cytochrome c, subunit IV mRNA is expressed at a surprisingly high level in testis. 3) While cytochrome c mRNA levels in liver are increased markedly in response to thyroid hormone treatment, subunit IV mRNA is not significantly affected. Differences in the expression of these two nuclear-encoded respiratory genes are consistent with differences in regulatory elements within their promoters. Therefore, the regulation of nuclear-encoded respiratory genes in response to tissue demands for cellular energy may not be satisfactorily explained by a set of universal regulators common to all such genes.

Animals↗
PubMed1990

NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells.

The assembly of the respiratory apparatus requires the coordinate expression of a large number of genes from both nuclear and mitochondrial genetic systems. In vertebrate organisms, the molecular mechanisms integrating the activities of these distinct genomic compartments in response to tissue demands for respiratory energy remain unknown. A potential inroad to this problem came with the discovery of nuclear respiratory factor 1 (NRF-1), a novel transcriptional activator defined by mutational and DNA binding analysis of the somatic cytochrome c promoter. Functional NRF-1 sites are now observed in several other recently isolated nuclear genes whose products function in the mitochondria. Among these are genes encoding subunits of the cytochrome c oxidase (subunit VIc) and reductase (ubiquinone-binding protein) complexes. In addition, a functional NRF-1 site resides in the MRP RNA gene encoding the RNA moiety of a ribonucleoprotein endonuclease involved in mitochondrial DNA replication. Synthetic oligomers of these sites competitively displace NRF-1 binding to the cytochrome c promoter. NRF-1-binding activities for each site also have the same thermal lability, copurify chromatographically, and make similar guanosine nucleotide contacts within each recognition sequence. Moreover, NRF-1 recognition in vitro correlates with the ability of each site to stimulate expression in vivo from a truncated cytochrome c promoter. The presence of NRF-1-binding sites in nuclear genes encoding structural components of the mammalian electron transport chain, as well as the mitochondrial DNA replication machinery, suggests a mechanism for coordination of nuclear and mitochondrial genetic systems through the concerted modulation of nuclear genes.

Animals↗
PubMed1990

In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins.

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.

Amino Acid Sequence↗
PubMed1990

Mitochondrial targeting of yeast apoiso-1-cytochrome c is mediated through functionally independent structural domains.

An iso-1-cytochrome c-chloramphenicol acetyltransferase fusion protein (iso-1/CAT) was expressed in Saccharomyces cerevisiae and used to delineate two stages in the cytochrome c import pathway in vivo (S. H. Nye and R. C. Scarpulla, Mol. Cell. Biol. 10:5753-5762, 1990 [this issue]). Fusion proteins with the CAT reporter domain in its native conformation were arrested at the initial stage of mitochondrial membrane recognition and insertion. In contrast, those with a deletional disruption of the CAT moiety were relieved of this block and allowed to translocate to the intermembrane space, where they functioned in respiratory electron transfer. In the present study, iso-1/CAT was used to map structural determinants in apoiso-1-cytochrome c involved in the initial step of targeting to the mitochondrial membrane. Carboxy-terminal deletions revealed that one of these determinants consisted of the amino-terminal 68 residues. Deletion mutations either within or at the ends of this determinant destroyed mitochondrial targeting activity, suggesting that functionally important information spans the length of this fragment. Disruption of an alpha-helix near the amino terminus by a helix-breaking proline substitution for leucine 14 also eliminated the targeting activity of the 1 to 68 determinant, suggesting a contribution from this structure. A second, functionally independent targeting determinant was found in the carboxy half of the apoprotein between residues 68 and 85. This determinant coincided with a stretch of 11 residues that are invariant in nearly 100 eucaryotic cytochromes c. Therefore, in lieu of an amino-terminal presequence, apocytochrome c has redundant structural information located in both the amino and carboxy halves of the molecule that can function independently to specify mitochondrial targeting and membrane insertion in vivo.

Apoproteins↗
PubMed1989

Introns in the 3'-untranslated region can inhibit chimeric CAT and beta-galactosidase gene expression.

The expression of a cyc::cat [cytochrome c/chloramphenicol acetyltransferase (CAT)] chimeric gene was stimulated 100-fold by the inclusion of a cyc intron in the 5'-untranslated region. In contrast, a single intron in the 3'-untranslated region was at best only slightly stimulatory, and surprisingly, inhibited expression of cat when an intron was also included in the 5'-untranslated region. This inhibition was independent of the identity of the downstream intron, occurring when either the simian virus 40 (SV40) small t intron or a cyc intron was located downstream from the cat coding region. Analysis of CAT mRNA levels, using a riboprobe spanning the 5' end of the CAT message, revealed that the stimulatory effect of a 5'-noncoding region intron were manifest at both the protein and RNA levels, whereas the inhibitory effects of 3'-noncoding region introns were detectable only at the protein level. The effects of intron position on chimeric gene expression were observed in both primate and rodent cell lines and also when the beta-galactosidase coding region was substituted for that of cat. Therefore, the common placement of an intron in the 3'-noncoding region is not the most beneficial to the expression of cyc chimeric genes. The position of introns within a transcriptional unit is a major factor to be considered when optimizing the efficiency of animal cell expression vectors.

Animals↗
PubMed1989

Interaction of nuclear factors with multiple sites in the somatic cytochrome c promoter. Characterization of upstream NRF-1, ATF, and intron Sp1 recognition sequences.

The rat somatic cytochrome c promoter is resolved into a mosaic of cis-acting upstream and intron elements required for maximal activity. Mutations in each diminished cytochrome c promoter activity and eliminated the specific binding of cognate nuclear factors. Among these is the recognition sequence for a nuclear factor designated NRF-1 (nuclear respiratory factor 1) also found in the upstream regions of several other nuclear genes whose products function in the mitochondria. The NRF-1 site was tightly coupled to a second functionally independent element (region I), and together these sites constitute a major determinant of cytochrome c expression. In addition to these novel sequence elements, the promoter also contained recognition sites for the common transcriptional activators ATF and Sp1. A potent promoter element within the first intron consisted of two adjacent Sp1 binding sites. Point mutations in the first site eliminated the promoter activity of the element as well as Sp1 binding to both sites. An ATF recognition sequence in the upstream promoter was identical to an authentic cyclic AMP (cAMP) responsive element in stimulating promoter activity and in conferring a cAMP response upon a heterologous promoter. These promoter elements and their cognate nuclear factors likely contribute to the housekeeping function of cytochrome c and to the coordinate modulation of respiratory gene expression according to cellular energy demands.

Animals↗
PubMed1988

Structure and expression of rodent genes encoding the testis-specific cytochrome c. Differences in gene structure and evolution between somatic and testicular variants.

Mammalian testis contains two forms of cytochrome c, one identical to the form found in somatic tissues and a second that is expressed in a stage-specific manner during spermatogenic differentiation. We have isolated both rat and mouse cDNA clones and the rat gene encoding the testis-specific cytochrome c and determined their DNA sequences. The testicular variant displays a number of notable differences with its somatic counterpart. 1) In contrast to the multipseudogene family derived from mammalian somatic cytochrome c genes, the testis gene is single-copy in genomic DNA with no detectable pseudogenes. 2) The rat testis gene is approximately 7 kilobases (kb) long with three introns totaling nearly 6.5 kb whereas the two introns dividing the 2.1-kb somatic gene occupy only 0.9 kb. Introns differ in position as well as size. 3) The testicular variant has a longer 5'-untranslated leader (230 versus 70 base pairs for the somatic gene) with an upstream open reading frame of 129 base pairs beginning with an AUG in a favorable translational context. 4) A single polyadenylation site in the testicular mRNA (approximately 900 nucleotides) contrasts with the three functionally equivalent sites observed in rat somatic messages. 5) Finally, rat and mouse testis cytochromes c differ at 4 amino acid residues as opposed to the complete sequence identity found in the somatic proteins suggesting a shorter unit evolutionary period for these molecules. These observations are consistent with a duplication of an ancestral cytochrome c gene leading to the emergence of novel structural features and regulatory properties likely associated with the striking tissue specificity of the testicular cytochrome c.

Amino Acid Sequence↗
PubMed1988

The human somatic cytochrome c gene: two classes of processed pseudogenes demarcate a period of rapid molecular evolution.

We have isolated and determined the DNA sequences of the human somatic cytochrome c gene (HCS) and 11 processed pseudogenes. HCS is the functional homologue to the previously characterized rat somatic gene because it correctly encodes the human heart protein, is present in single copy in the human genome, is nearly identical in both size and intron/exon structure to rodent somatic genes, and shares a high degree of sequence homology with its rat counterpart including a well-conserved promoter region (77% over 250 nucleotides). In contrast to the rodent system, however, where the known pseudogenes all originated from a locus encoding the present day cytochrome c, the human pseudogenes are of two types. A predominant class of older pseudogenes came from a progenitor of HCS that encoded an ancestral form of the protein, while a second group of only a few young pseudogenes originated from a recent parent of HCS that encoded the current cytochrome c polypeptide. These two distinct classes of human pseudogenes provide a molecular record of the history of cytochrome c evolution in primates and demarcate a short period of rapid evolution of the functional gene.

Animals↗
PubMed1988

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

Animals↗
PubMed1986

Coordinate induction of multiple cytochrome c mRNAs in response to thyroid hormone.

Because of the well documented influence of thyroid hormones in the general control of oxidative metabolism and their putative role in the transcriptional activation of specific genes, changes in the levels of multiple cytochrome c mRNAs were monitored in thyroidectomized rats after administration of 3,5,3'-triiodo-L-thyronine. In contrast to normal animals where the concentration of cytochrome c mRNAs in the polyadenylated RNA fraction is approximately 4- to 5-fold higher in kidney than in liver, hypothyroid animals displayed an equivalent low level of all three mRNAs (1400, 1100, and 700 nucleotides) in both tissues. Following the establishment of chronic hyperthyroidism, the levels of the three mRNAs were coordinately elevated by about 4- to 5-fold resulting in their restoration to approximately normal amounts for kidney but to a level substantially above the normal for liver. Only modest induction of mRNA is detected in the first 12 h following a single intravenous injection of 3,5,3'-triiodo-L-thyronine. The major increase occurs between 12 and 24 h with the plateau level attained between 24 and 48 h. The magnitude of the response is in excess of the general increase in total cellular RNA mediated by the hormone. Induction of the cytochrome c mRNAs is coincident with an elevation in gene transcription of comparable magnitude detected using nascent RNA chains synthesized by isolated nuclei. The kinetics of these responses are similar to those observed for the increase in respiratory activity mediated by the hormone.

Animals↗
PubMed1986

Evolution of cytochrome c genes and pseudogenes.

A statistical analysis of the nucleotide sequences of cytochrome c genes from four species of animals and two of yeast and of cytochrome c pseudogenes from rat, mouse, and human was conducted. It was estimated that animals and yeast diverged 1.2 billion years ago, that the two duplicated genes DC3 and DC4 in Drosophila diverged 520 million years ago, and that the two duplicated genes Iso-1 and Iso-2 in the yeast Saccharomyces cerevisiae diverged 200 million years ago. DC3 is expressed at a low level and has evolved 3 times faster than DC4. This observation supports the neutralist view that relaxation of functional constraints is a more likely cause of accelerated evolution following gene duplication than is advantageous mutation. All the rodent pseudogenes examined appear to be processed pseudogenes derived directly from the functional genes, and most of them apparently arose after the mouse-rat split. No event of gene conversion could be detected between any pair of the rodent pseudogenes. Our analysis suggests that the human cytochrome c gene has evolved at a rate comparable to the average rate for pseudogenes, whereas some human cytochrome c pseudogenes have evolved at an exceptionally low rate.

Animals↗
PubMed1986

Functional expression of rat cytochrome c in Saccharomyces cerevisiae.

To determine whether a mammalian cytochrome c could efficiently replace iso-1-cytochrome c, which is encoded by the yeast CYC1 gene, the coding sequence of RC9 (a nondefective processed gene from rat) was cloned in both single- and multiple-copy expression vectors under the direction of the yeast alcohol dehydrogenase 1 (ADC1) promoter. Upon transformation of a CYC1 deletion strain, the multiple-copy construct restored a wild-type growth rate on lactate medium; such growth normally requires derepressed amounts of iso-1-cytochrome c. These transformants expressed a level of hybrid ADC1/RC9 mRNA approximately 5- to 10-fold greater than the amount of message from the endogenous ADC1 gene and produced a steady-state level of rat cytochrome c equivalent to that of the wild-type yeast protein. A requirement for the vector was evidenced by its absence in all transformants that lost the lactate growth phenotype after propagation in nonselective medium. In contrast, the level of vector-specific message in single copy was equivalent to that of the endogenous ADC1 mRNA, but transformants exhibited no significant growth on lactate. Constructions having a small deletion or a mammalian intron within the rat cytochrome c coding region failed to support lactate-dependent growth, indicating that complementation depends upon proper translation of the correct rat coding sequence. Therefore, the rat polypeptide, when expressed at normal physiological levels, is recognized by the yeast machinery involved in the multiple steps required for the processing and transport of an active cytochrome c as well as its functional interaction with the respiratory apparatus.

Animals↗
PubMed1985

Association of a truncated cytochrome c processed pseudogene with a similarly truncated member from a long interspersed repeat family of rat.

The cytochrome c multigene family of rat contains approximately 30 processed pseudogenes that represent genomic DNA copies of three alternate mRNAs. Here, the DNA sequence of an unusual processed pseudogene reveals that it has a complete 3' noncoding region including a short poly A tail but unlike the others is abruptly truncated at its 5' end, 19 amino acid codons from the translation terminator. At this position the pseudogene is fused through 17 consecutive adenylic acid residues to a 1.3 kb repetitive sequence. This repetitive element is flanked by direct repeats and represents a truncated member from a major long interspersed repeat family. The rat element is a composite of sequences observed in long interspersed repeats from both rodents and primates. Comparison to the equivalent mouse sequences shows that the 5' half of the repeat distal to the pseudogene has an open reading frame and is highly conserved whereas the half adjacent to the pseudogene is evolutionarily unstable. The proportion of cytochrome c pseudogene recombinant clones containing this repetitive DNA is 3 fold greater than observed in random isolates and may reflect a general tendency of processed pseudogenes to associate with other repetitive sequences in the genome.

Animals↗
PubMed1984

Processed pseudogenes for rat cytochrome c are preferentially derived from one of three alternate mRNAs.

Three cytochrome c mRNAs (1,400, 1,100 and 700 nucleotides) are colinear with RC4, a gene that has introns and correctly encodes cytochrome c. A comparison of RC4 to six nonallelic clones isolated from the rat cytochrome c multigene family demonstrates that all three mRNAs are represented in the genome as processed pseudogenes. Four of the six pseudogenes are derived from the 1,100-nucleotide mRNA, and genomic hybridizations further establish that nearly all of the 30 or so gene family members are also genomic copies of this mRNA despite the equimolar ratio of the three messages in rat tissues. Thus, the surprising multiplicity of cytochrome c sequences in the rat genome is mainly accounted for by the selective use of the 1,100-nucleotide mRNA for the formation of processed pseudogenes. In contrast to 700- and 1,400-nucleotide species which are polyadenylated downstream from AAGUAAA and AAUUAAA, respectively, the 1,100-nucleotide mRNA uses the ubiquitous AAUAAA and also displays a unique stem and loop structure (delta G = -59.4 kJ) centered 37 base pairs upstream from this sequence.

Animals↗
PubMed1983

Nonallelic members of the cytochrome c multigene family of the rat may arise through different messenger RNAs.

We determined the nucleotide sequences of three nonallelic cytochrome c genes (from recombinant clones Ch4A-RC5, 6 and 8) isolated from the rat cytochrome c gene family. In contrast with a fourth gene (from Ch4A-RC4), which has an intron and correctly encodes rat cytochrome c, these three appear to be pseudogenes and resemble mRNA molecules in two respects: they are all missing the intron of clone 4, and sequence homology with clone 4 in their 3' noncoding regions abruptly ends at two different A-rich tracts reminiscent of poly(A) tails. We also detect three cytochrome c mRNAs of sizes 1400, 1100 and 700 nucleotides in several tissues of the adult rat. The size differences among the mRNAs can be accounted for by length heterogeneity in their 3' noncoding regions. Two of the 3' ends map to the two points where the mRNA-like genes diverge from clone 4 at poly(A) tracts. Furthermore, short direct repeats flank the genes of clones 5, 6 and 8 at the positions where their sequences diverge. The observations suggest that these members of the cytochrome c multigene family may arise through insertion into the genome of DNA copies of cytochrome c mRNAs.

Animals↗