The elucidation of the human mitochondrial genome: a historical perspective.
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DNA, Mitochondrial↗
Biomedical subjects
G Attardi
Publications and source records attributed to G Attardi.
Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.
In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.
Antibodies↗
Seven unidentified reading frames of human mitochondrial DNA encode subunits of the respiratory chain NADH dehydrogenase.
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Cytochrome Reductases↗
Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.
A ribonuclease P-like activity was partially purified from HeLa cell mitochondria by DEAE-cellulose and octyl-Sepharose chromatography. RNase P-like activity can be quantitatively recovered from intact mitochondrial preparations treated with micrococcal nuclease, strongly suggesting that the enzyme is localized within the organelles. Mitochondrial RNase P (mtRNase P) cleaves the precursor to Escherichia coli suppressor tRNATyr at the same site as E. coli RNase P, producing the mature 5'-end of tRNATyr. The sensitivity of mtRNase P to pretreatment with nucleases or Pronase indicates that the enzyme has essential RNA and protein components. Although the ionic requirements of mtRNase P are similar to those of the RNase P activity isolated from the post-mitochondrial cytosol fraction, the chromatographic properties of mtRNase P are distinct. Mitochondrial RNase P is probably a part of the mitochondrial RNA processing machinery of mammalian mitochondria, being responsible for the endonucleolytic cleavage of the RNA transcripts at the 5'-side of the tRNA sequences.
Adenosine Triphosphate↗
Characterization of an RNA polymerase activity from HeLa cell mitochondria, which initiates transcription at the heavy strand rRNA promoter and the light strand promoter in human mitochondrial DNA.
An RNA polymerase activity capable of initiating transcription at both the heavy strand rRNA promoter and the light strand promoter of human mitochondrial DNA has been partially purified from HeLa cell mitochondria and characterized in its requirements and products. The ratio of the two transcription initiating activities varied considerably from preparation to preparation. The human mtRNA polymerase partially purified by DEAE-cellulose and heparin-agarose chromatography exhibits a great sensitivity to ionic strength and to Mn2+, characteristics which clearly differentiate this enzyme from bacterial and eukaryotic nuclear RNA polymerases, and in contrast resemble the behavior of the yeast mtRNA polymerase. The human mtRNA polymerase exhibits a requirement for ATP which is 15- to 20-fold higher than that for the other NTPs, a low optimum template DNA concentration, and a marked susceptibility to inhibition by non-mitochondrial DNA.
Adenosine Triphosphate↗
Assignment of human dihydrofolate reductase gene to band q23 of chromosome 5 and of related pseudogene psi HD1 to chromosome 3.
The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a number of human-Chinese hamster ovary cell hybrids containing terminal and interstitial deletions in human chromosome 5. A correlation of genomic blotting data with the chromosome 5 constitution of the individual hybrids has allowed the assignment of the human DHFR gene to 5q23. The present work also establishes the location of the related intronless pseudogene psi HD1 in chromosome 3.
Animals↗
Animal mitochondrial DNA: an extreme example of genetic economy.
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Amino Acid Sequence↗
Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.
The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and S1 protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1- and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.
Base Sequence↗
Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes.
The complete human dihydrofolate reductase (DHFR) gene has been cloned from four recombinant lambda libraries constructed with the DNA from a methotrexate-resistant human cell line with amplified DHFR genes. The detailed organization of the gene has been determined by restriction mapping of the cloned fragments and DNA sequencing of all the protein coding regions and adjacent intron segments, and shown to correspond to that of the native human DHFR gene. The gene spans a length of approximately 29 X 10(3) bases from the ATG initiator codon to the end of the 3' untranslated region, and contains five introns that interrupt the protein coding sequence. The number and positions of introns are identical to those found in the mouse gene. By contrast, the size of the homologous introns (with the exception of the first one) varies greatly, up to several fold, in the genes from man, mouse and Chinese hamster; the intron sequences also exhibit a great divergence, except in the junction regions. A striking sequence homology, extending over several hundred nucleotides, exists between the human and mouse gene 5' non-coding regions. These regions are characterized by an unusually high G + C content, 72% and 66% in the human and mouse genes, respectively, which is maintained in the first coding segment and first intron, and is in sharp contrast to the relatively low G + C content (approximately 40%) of the remainder of the gene.
Animals↗
Intercalating drugs and low temperatures inhibit synthesis and processing of ribosomal RNA in isolated human mitochondria.
Mitochondrial DNA transcription in isolated HeLa cell mitochondria faithfully reproduces the in vivo process. In this system, actinomycin D, proflavine and ethidium bromide preferentially inhibit the formation of ribosomal RNA over that of messenger RNA, strongly supporting independent controls of the two overlapping transcription units involved in their synthesis. The processing step removing the tRNAPhe sequence at the 5' end of the ribosomal RNA precursor is uniquely sensitive to low temperature, proflavine and ethidium bromide.
DNA, Mitochondrial↗
A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments.
Several clones of rDNA have been isolated from an adult human liver DNA Charon 4A library by using cDNA probes synthesized from human 18S and 28S rRNA. The insert of one recombinant Charon 4A clone contained, besides the already known 5.7-kb EcoRI fragment of rDNA, comprising the major portion of the 18S rRNA gene and all the external transcribed spacer (ETS), a previously unidentified EcoRI fragment of rDNA of 8.5 kb in size. DNA transfer hybridization experiments utilizing EcoRI digests of the human DNA used to construct the library and of another human DNA showed the presence of the 8.5-kb EcoRI fragment in a minority of the rDNA repeats on the 5'-end side of the 5.7-kb fragment, thus defining a hitherto unidentified type of EcoRI polymorphism of these repeats.
Adult↗
Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes.
The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a large number of human-mouse cell hybrids containing overlapping subsets of human chromosomes. A correlation of genomic blotting data with the chromosome constitution of the individual cell hybrids has allowed the assignment of the human DHFR gene to chromosome 5. This chromosome assignment has been confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin, a marker associated with chromosome 5, in two human-mouse cell hybrids selected for resistance to the toxin. Six EcoRI fragments of human DNA containing DHFR pseudogenes or other DHFR-related sequences have been assigned to chromosomes other than chromosome 5.
Animals↗
Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.
A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.
DNA Restriction Enzymes↗
A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA.
The presence of dihydrofolate reductase (DHFRase)-specific sequences that, in contrast to the normal DHFRase gene, are not amplified in a methotrexate-resistant cell line, has been detected in the DNA from human sperm and from several human cell lines. DNA fragments containing some of these sequences have been isolated from a cosmid library of human sperm DNA. One of these fragments contains a DHFRase pseudogene (psi HD1) that completely lacks introns, has 92% sequence homology to the corresponding region of normal DHFRase complementary DNA, but exhibits several alterations that make it nonfunctional. The sequence analysis of the inserts of four different plasmids containing the reading frame and varying lengths of the 3' non-coding regions of human DHFRase-specific cDNAs has revealed that the 3' non-coding segments all are colinear in their corresponding portions. Furthermore, the data indicate that the cDNA of one of the plasmids is probably derived from the smallest of the three main human DHFRase messenger RNAs, the 0.8 X 10(3) base (0.8 kb) mRNA, the cDNA of two others, from the 1.0 kb mRNA, and the cDNA of the fourth, from a longer mRNA. These results are consistent with the idea that the multiple forms of DHFRase mRNA in human cells derive from the same gene by different transcription or RNA-processing events. Moreover, the sequence comparison between the psi HD1 and the different DHFRase cDNAs clearly indicates that, if an mRNA intermediate has participated in the formation of this pseudogene, a form of mRNA larger than the 1.0 kb mRNA, probably the 3.8 kb mRNA, must have been involved.
Base Sequence↗
Analysis of human mitochondrial RNA.
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Base Sequence↗
The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units.
A detailed analysis of the mapping and kinetic properties of oligo(dT)-cellulose bound and unbound transcripts synthesized in HeLa cells has indicated that two distinct transcription events take place in the rDNA region. Of these, one appears to start approximately 25 bp upstream of the tRNAPhe gene and to terminate at or near the 3' end of the 16S rRNA gene, being responsible for the synthesis of the bulk of rRNA. The other transcription event starts near the 5' end of the 12S rRNA gene, proceeds beyond the 3' end of the 16S rRNA gene, and results in the synthesis of a polycistronic molecule corresponding to almost the entire H-strand, which is destined to be processed to yield the mRNAs and most of the tRNAs encoded in the heavy strand. The existence of two overlapping transcription units with distinct promoters is probably the basis for the differential regulation of synthesis of the rRNAs and heavy-strand-coded mRNAs.
DNA↗
Antibodies against synthetic peptides reveal that the unidentified reading frame A6L, overlapping the ATPase 6 gene, is expressed in human mitochondria.
Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to detect human mitochondrial gene products. In particular, antibodies directed against either the NH2-terminal decapeptide or the COOH-terminal undecapeptide of cytochrome c oxidase subunit II (COII) were both very effective in immunoprecipitating the previously identified COII polypeptide from an SDS lysate of mitochondria from HeLa cells. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the unidentified reading frame A6L, which overlaps the ATPase 6 gene, immunoprecipitated specifically a component (#25) of the HeLa cell mitochondrial translation products; antibodies directed against the NH2-terminal octapeptide also precipitated protein 25, although less efficiently. The size of protein 25, as estimated from its electrophoretic mobility, is compatible with its being the unidentified reading frame A6L product. Furthermore, a fingerprinting analysis of this protein after trypsin digestion has given results consistent with this identification.
Adenosine Triphosphatases↗
Identification of the polypeptides encoded in the ATPase 6 gene and in the unassigned reading frames 1 and 3 of human mtDNA.
Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to identify the polypeptides encoded in the ATPase 6 gene and in unidentified reading frames (URFs) 1 and 3 of human mtDNA. In particular, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the ATPase 6 reading frame immunoprecipitated specifically component 17 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal undecapeptide of the putative URF1 product or against the COOH-terminal heptapeptide of the presumptive URF3 product were effective in immunoprecipitating specifically component 12 or, respectively, component 24 of the mitochondrial translation products. The sizes of proteins 17, 12, and 24, as estimated from their electrophoretic mobilities, are compatible with their being the products of the ATPase 6 gene, URF1, and URF3, respectively.
Adenosine Triphosphatases↗