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D K Hanson

Publications and source records attributed to D K Hanson.

31 records · Page 2Linked to original sources
PubMed1992

In bacterial reaction centers protons can diffuse to the secondary quinone by alternative pathways.

The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobacter capsulatus. In the L212-213AA double mutant (L212Glu----Ala, L213Asp----Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third 'compensating' mutation, M231Arg----Leu, at about 15 A from the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent pK values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to QB in Rb. capsulatus. We suggest that protons can be delivered to the QB site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center.

Binding Sites↗
PubMed1992

Femtosecond spontaneous-emission studies of reaction centers from photosynthetic bacteria.

Spontaneous emission from reaction centers of photosynthetic bacteria has been recorded with a time resolution of 50 fs. Excitation was made directly into both the special-pair band (850 nm) and the Qx band of bacteriochlorophylls (608 nm). Rhodobacter sphaeroides R26, Rhodobacter capsulatus wild type, and four mutants of Rb. capsulatus were studied. In all cases the fluorescence decay was not single exponential and was well fit as a sum of two exponential decay components. The short components are in excellent agreement with the single component detected by measurements of stimulated emission. The origin of the nonexponential decay is discussed in terms of heterogeneity, the kinetic scheme, and the possibility of slow vibrational relaxation.

Energy Transfer↗
PubMed1986

Mitochondrial genome rearrangement leads to extension and relocation of the cytochrome c oxidase subunit I gene in sorghum.

The mitochondrial gene (COXI) encoding cytochrome c oxidase subunit I (COI) was isolated from two cytoplasmic genotypes of sorghum that synthesize different COI polypeptides. The Milo COI (Mr approximately 38,000) is encoded by a 530 codon structural gene sharing 98% homology with the corresponding maize gene. A variant COI observed in 9E cytoplasm (Mr approximately 42,000) is encoded by a 631 codon structural gene that diverges completely from the Milo COXI gene both 100 bp 5' to the presumed initiator methionine and within the 3' coding sequence. The 3' divergence results in a 101 C-terminal extension of the 9E COI that is not homologous to any known mitochondrial polypeptide. The novel 9E COXI, apparently arising from at least two rearrangements, affects transcription and gene product.

Base Sequence↗
PubMed1983

Functional domains in introns. RNA processing intermediates in cis- and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b.

The penultimate intron of the split mitochondrial gene (cob) for apocytochrome b of Saccharomyces cerevisiae is of particular interest; it contains a long unassigned reading frame, is present in both long form (six exons) and short form (three exons) of the gene, and a product expressed from it is required for the removal of its transcript and that of an intron in the transcript of the oxi3 gene. Complementation analysis shows mutants in this intron to be either cis-dominant or transrecessive. Cis-dominant mutants are located in the first third (approximately 350 base pairs) of the open and near the 3'-end of the closed reading frame, while trans-recessive mutants are scattered throughout the remaining two-thirds (approximately 750 base pairs) of the open frame. Mutants in both classes exhibit the same pattern of splicing defects in their transcripts, but for different reasons. Those in the trans-recessive class lack a functional maturase (probably a protein of Mr = 27,000) encoded wholly within the 3'-terminal segment of the intron, and for this reason also fail to express oxi3. In contrast, cis-dominant mutants are incapable of providing the splicing complex with a substrate of appropriate 2 degrees structure. They also accumulate a novel transcript, 1900 nucleotides long, which contains the intron fused to the downstream (3') exons. This may reflect an inability of the splicing complex to complete the normal sequence of cleavage of the intron at its downstream junction and the ligation of the two exonic moieties.

Base Composition↗
PubMed1982

Evidence for translated intervening sequences in the mitochondrial genome of Saccharomyces cerevisiae.

In yeast, the mitochondrial genes for subunit I of cytochrome oxidase (oxi3) and for apocytochrome b (cob) are known to be split. In some strains, the latter contains five intervening sequences, three of which coincide with clusters of mutational sites referred to in their order of transcription as the loci box3, 10, and 7, respectively. Mutations at the first of these result in the accumulation of novel, large polypeptides (apparent Mr congruent to 43,000) believed to originate from a fusion of sequences found in the NH2-terminal segment of apocytochrome b to others encoded in the intervening sequence itself. We now provide evidence for close similarities of at least a part of translated intron sequences between (a) mutants in box7 in "long" form and "short" form strains (which lack the first three introns including the one for the box3 locus); (b) mutants in a subset of box7 mutants and those in box3, and (c) between intron sequences in box7 and a sequence presumably encoded in oxi3. These structural homologies have been analyzed and shown to be referable to sequence homologies in two proteins, one derived from the second intron (box3) in cob and the other from oxi3. The accumulation in certain cob mutants of proteins and of a transcript containing a sequence specified by oxi3 provides additional strong evidence for the previously suggested regulation of oxi3 by the penultimate, box7-containing intron of cob.

Base Sequence↗
PubMed1982

Functional domains in introns: trans-acting and cis-acting regions of intron 4 of the cob gene.

We have sequenced the mutational changes in eight mutants in the open reading frame of intron 4 of the cob gene on yeast mitochondrial DNA. Three have a cis-acting splicing defect, while the other inactivate a trans-recessive intron domain that specifies a trans-acting splicing factor. From phenotypic evidence, including analyses of the allele-specific extra proteins, we have identified a protein (P27) encoded wholly within the intron that appears to be the intron 4 splicing factor (maturase). The evidence suggests that P27 is a secondary translation product resulting from the proteolytic cleavage of a larger precursor encoded by exon and intron sequences, but an alternative model, in which P27 is a primary translation product, has not been ruled out.

Amino Acid Sequence↗
PubMed1981

Regulatory interactions between mitochondrial genes: interactions between two mosaic genes.

We have studied the mitochondrial DNA and the phenotypes of strains of Saccharomyces cerevisiae with specific intervening sequences in two mosaic genes: cob (the gene for apocytochrome b) and oxi3 (the gene for subunit I of cytochrome oxidase). The results suggest the following. (i) The presence of an intervening sequence downstream encompassing the intron box7 is sufficient for the regulation of oxi3 by cob (BOX phenotype); two sequences (containing intron loci box3 and box10) upstream in cob and two in oxi3 are dispensable. (ii) Strains without the two sequences upstream still contain the downstream sequence and the competence to specify a functional trans-acting element. Mutational lesions in this segment are phenotypically indistinguishable from box7 mutants, including the accumulation of polypeptides with homologous amino acid sequences. (iii) A catabolite-sensitive BOX phenotype, characteristic of mutants in the first exon, requires the simultaneous presence of an adjacent intervening sequence. A model is presented in which a hypothetical product specified by an intron (locus box7) of the cob gene controls the expression of a second mosaic gene (oxi3).

DNA Restriction Enzymes↗
PubMed1980

Mosaic organization of a mitochondrial gene: evidence from double mutants in the cytochrome b region of Saccharomyces cerevisiae.

The region coding for apocytochrome b in the mitochondrial genome of Saccharomyces cerevisiae is believed to exhibit a mosaic organization, consisting in certain strains of five exons and four introns. This model can be tested by the use of double mutants, each containing two physically, genetically and phenotypically defined mit- lesions in cis, (that is, in the same mitochondrial chromosome). Such mutants have been constructed, and the phenotypes of several examples of each of the four possible classes--exon-exon, exon-intron (downstream), intron (upstream)-exon and intron-intron--have been examined. Our results have shown that upstream mutations are always epistatic to downstream ones for polypeptide products, and that regulation of expression of cytochrome oxidase subunit I by introns is epistatic regardless of position. These findings have provided an independent verification of the mosaic model, and also suggest that at least the majority of novel polypeptides accumulating in intron mutants are hybrid products that contain sequences of the wild-type polypeptide.

Apoproteins↗
PubMed1979

Regulatory interactions between mitochondrial genes. I. Genetic and biochemical characterization of some mutant types affecting apocytochrome b and cytochrome oxidase.

The region of the mtDNA containing the structural gene for apocytochrome b is called the cob or box region. There is evidence that the same region is also involved in the regulation of cytochrome oxidase. We have isolated eight mit- mutants in this region and have ordered them using petite deletion mapping. Four of these mutants appear to map outside the boxII region on the oli2-proximal end. Analysis of restriction endonuclease fragments of the mtDNA from peptides used in the deletion mapping suggests a minimum size of 3.1 x 10(3) base pairs for the whole cob region. Although none of our mutants contained any cytochrome b or cytochrome b-linked activities, polypeptides apparently related to apocytochrome b were present in some but not all mutants. Additional regulatory effects (both positive and negative) on cytochrome oxidase by virtue of control of its subunit I were also observed. In addition to these phenotypic traits, some of the mutants accumulated novel, mitochondrially translated polypeptides not seen in wild type.

Apoproteins↗
PubMed1979

Regulatory interaction between mitochondrial genes. II. Detailed characterization of novel mutants mapping within one cluster in the cob2 region.

These studies describe the properties of three mit- mutants designated EM17, EM25, and PZ1, all mapping at two closely linked sites near one of the boundaries of the region of the mitochondrial genome concerned with the specification of cytochrome b. They all exhibit complex phenotypes affecting cytochrome b, cytochrome aa3, and additional polypeptides not found in the wild type. In the case of EM 17 this complexity can be ascribed to the presence of two mutations induced in the course of the initial mutagenic treatment: one, the cob2 mutation proper, is responsible for the loss of cytochrome b which is replaced by an altered, functionally inactive polypeptide, cytochrome b. This polypeptide can be further modified, or even eliminated, by the controlled introduction of another mutation in the cob1 segment of the cob region. The reduction in cytochrome oxidase subunit I, responsible for the effects on cytochrome aa3 and enzymatic activity in EM17, is due to a second (not mit-) mutation that has been located in the par1-proximal segment of the oxi3 region. This second mutation as well as the cob mutation can be overcome, and the respective aspect of wild type function restored to EM17, by recombination with rho- strains retaining the appropriate segment(s) of the wild type genome. The phenotype of the other two mutants is due to a single mutagenic event. This conclusion is confirmed by their ability to restore wild type functions by reversion. The mutation in EM25 appears to be due to a frameshift, which has led to premature chain termination, producing a polypeptide of Mr = 15,000 related to apocytochrome b. This change is accompanied by a decrease in the amount of subunit I of cytochrome oxidase. Revertants fall into three classes: on galactose two produce a polypeptide indistinguishable from apocytochrome b, but vary in its amount, while the third fails to increase apocytochrome b above mutant levels. Production of subunit I is increased but fails to reach wild type levels. Complete restoration of wild type functions can, however, be obtained by recombination of EM25 with rho- (cob2+) strains. Mutation PZ1 results in a complete absence of any polypeptide related to apocytochrome b and of cytochrome oxidase subunit I. These cells produce a novel polypeptide with a Mr = 45,000 not found in the wild type, and unrelated to all its normal polypeptides. Reversion or recombination with rho- (cob2+) strains results in virtually complete restoration of all wild type functions and the elimination of the novel polypeptide.

Apoproteins↗