Biomedical subjects

H Janska

Publications and source records attributed to H Janska.

8 recordsLinked to original sources

PubMed2001

The origin and maintenance of the small repeat in the bean mitochondrial genome.

The genesis of small repeats involved in infrequent recombinations in plant mitochondrial genomes remains unclear. We propose that at least some of the small repeats are generated in a similar way to the large, highly recombinogenic, plant mitochondrial repeats. A 314-bp sequence was detected as a small, rarely recombining mitochondrial repeat in the genus Phaseolus. Two of the recombinational forms were predominant, while two others were found in substoichiometric amounts in the species P. vulgaris, P. polyanthus and P. coccineus. However, the pairs of predominant and substoichiometric forms were distinct in each genome, indicating that a mechanism other than recombination is responsible for their maintenance in high or low copy number. In P. lunatus, which is phylogenetically quite remote from the other species examined, only one form of the 314-bp repeat was predominant, while the other forms were present in substoichiometric amounts. In this genome, we also identified sequences containing the terminal 11 or 7 bp of the 314-bp repeat. These configurations could serve as intermediates during generation of the repeat. We presume that two site-specific recombinations between the intermediates and the predominant form found in P. lunatus resulted in creation of the two new forms of the repeat. The fourth form of the repeat appeared after a further recombination that occurred at the substoichiometric level. The nature of this recombination, whether site-specific or homologous, is discussed. Beyond the evident similarities between the model presented in this work and the three-recombination models previously proposed to explain the formation of large, frequently recombining repeats, we did not detect any specific deletion associated with generation of the repeat.

Base Sequence↗

PubMed2001

Tracing evolutionary and developmental implications of mitochondrial stoichiometric shifting in the common bean.

The recombination and copy number shifting activities of the plant mitochondrial genome are widely documented across plant genera, but these genome processes have not been as well examined with regard to their roles in plant evolution. Because of the extensive plant collections of Phaseolus spp and the degree to which cytoplasmic male sterility (cms) has been characterized in the common bean, this system would be valuable for investigating mitochondrial genome dynamics in natural populations. We have used the cms-associated sequence pvs-orf239 as a mitochondrial genetic marker for these studies and have demonstrated its universal presence throughout a diversity of undomesticated Phaseolus lines. Within these populations, the pvs-orf239 sequence is present in high copy number in approximately 10% of the lines, but substoichiometric in all others. This mitochondrial sequence, derived apparently by at least two recombination events, is well conserved with two point mutations identified that are both apparently silent with regard to the sterility phenotype. A putative progenitor sequence was identified in Phaseolus glabelus in substoichiometric levels, suggesting that the present-day pvs-orf239 sequence was likely introduced substoichiometrically. Copy number shifting within the mitochondrial genome results in a 1000- to 2000-fold change, so that substoichiometric forms are estimated at less than one copy per every 100 cells. On the basis of PCR analysis of root tips, we postulate that a mitochondrial "transmitted form" resides within the meristem to assure transmission of a complete genetic complement to progeny.

Cloning, Molecular↗

PubMed1998

Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility

The plant mitochondrial genome is characterized by a complex, multipartite structure. In cytoplasmic male-sterile (CMS) common bean, the sterility-inducing mitochondrial configuration maps as three autonomous DNA molecules, one containing the sterility-associated sequence pvs-or f 239. We constructed a physical map of the mitochondrial genome from the direct progenitors to the CMS cytoplasm and have shown that it maps as a single, circular master configuration. With long-exposure autoradiography of DNA gel blots and polymerase chain reaction analysis, we demonstrate that the three-molecule CMS-associated configuration was present at unusually low copy number within the progenitor genome and that the progenitor form was present substoichiometrically within the genome of the CMS line. Furthermore, upon spontaneous reversion to fertility, the progenitor genomic configuration as well as the molecule containing the pvs-or f 239 sterility-associated sequence were both maintained at substoichiometric levels within the revertant genome. In vitro mitochondrial incubation results demonstrated that the genomic shift of the pvs-or f 239-containing molecule to substoichiometric levels upon spontaneous reversion was a reversible phenomenon. Moreover, we demonstrate that substoichiometric forms, apparently silent with regard to gene expression, are transcriptionally and translationally active once amplified. Thus, copy number suppression may serve as an effective means of regulating gene expression in plant mitochondria.

Journal Article↗

PubMed1993

Unusual mitochondrial genome organization in cytoplasmic male sterile common bean and the nature of cytoplasmic reversion to fertility.

Spontaneous reversion to pollen fertility and fertility restoration by the nuclear gene Fr in cytoplasmic male sterile common bean (Phaseolus vulgaris L.) are associated with the loss of a large portion of the mitochondrial genome. To understand better the molecular events responsible for this DNA loss, we have constructed a physical map of the mitochondrial genome of a stable fertile revertant line, WPR-3, and the cytoplasmic male sterile line (CMS-Sprite) from which it was derived. This involved a cosmid clone walking strategy with comparative DNA gel blot hybridizations. Mapping data suggested that the simplest model for the structure of the CMS-Sprite genome consists of three autonomous chromosomes differing only in short, unique regions. The unique region contained on one of these chromosomes is the male sterility-associated 3-kb sequence designated pvs. Based on genomic environments surrounding repeated sequences, we predict that chromosomes can undergo intra- and intermolecular recombination. The mitochondrial genome of the revertant line appeared to contain only two of the three chromosomes; the region containing the pvs sequence was absent. Therefore, the process of spontaneous cytoplasmic reversion to fertility likely involves the disappearance of an entire mitochondrial chromosome. This model is supported by the fact that we detected no evidence of recombination, excision or deletion events within the revertant genome that could account for the loss of a large segment of mitochondrial DNA.

Chromosome Mapping↗

PubMed1991

The amino-terminal sequence of the catalytic subunit of bovine enterokinase.

Bovine enterokinase (enteropeptidase) is a serine protease and functions as the physiological activator of trypsinogen. The enzyme has a heavy chain (115 kD) covalently linked to a light or catalytic subunit (35 kD). The amino acid composition showed that the light chain has nine half-cystine residues (four as intramolecular disulfides) and that one half-cystine was in a disulfide link between the light and heavy subunits. The amino-terminal 27 residues of the S-vinylpyridyl derivative of the light chain were determined by gas-phase Edman degradation. The sequence has homologies with other serine proteases containing one or two chains. The homologies suggest that the catalytic subunit has the same three-dimensional structure and, therefore, the same mechanism of enzymatic action as pancreatic chymotrypsin, trypsin, and elastase. The presence of the conserved amino-terminal activation peptide sequence (IVGG) shows that enterokinase must have a zymogen precursor and that the two-chain enzyme arises from limited proteolysis during posttranslational processing.

Amino Acid Sequence↗

PubMed1989

A direct high-performance liquid chromatography assay of the enzymatic activity of enterokinase (enteropeptidase).

Bovine enterokinase (enteropeptidase) activates trypsinogen to trypsin at pH 8.0. In the presence of chicken ovomucoid, a stable complex of ovomucoid-trypsin is produced, inactivating trypsin and eliminating autoactivation of trypsinogen. The molecular size of trypsin (24,000 Da) is increased twofold on forming the ovomucoid-trypsin complex (52,000 Da). Size-exclusion chromatography on a Toya Soda TSK G2000SW column in an HPLC system and with computer-assisted analyses gives a direct quantitative determination of the amount of substrate (trypsinogen) and product (ovomucoid-trypsin). The rate of disappearance of substrate is equal to the rate of formation of product in agreement with kinetic theory. The simultaneous determination of both rates increases the reliability of the assay. The HPLC assay has an extended linear range for the velocity of the activation process as a function of enzyme concentration. The assay is reliable and accurate for highly purified preparations, samples at different steps in the purification scheme, and for a direct assay of the intestinal contents. The assay should be useful in clinical analyses.

Animals↗

PubMed1989

Enterokinase (enteropeptidase): comparative aspects.

The serine protease enterokinase is the physiological activator of trypsinogen and has a specificity for the sequence (Asp)4-Lys-Ile. The enzyme consists of two subunits linked by a disulfide bond. The heavy chain achors enterokinase in the intestinal brush border membrane and the light chain is the catalytic subunit, which has the same mechanism of action as trypsin and chymotrypsin. Many properties of enterokinase resemble blood-clotting enzymes, suggesting that enterokinase lies on the same phylogenetic branch as the blood-clotting proteins.

Animals↗

PubMed1986

Catfish liver acid phosphatases: differently glycosylated enzyme molecules with altered kinetic properties.

Two forms of catfish liver acid phosphatase (AcPase I and II) were separated and purified to homogeneity and their carbohydrate compositions and some biochemical properties were studied. Evidence is given that AcPase I and II are differently glycosylated forms of the same enzyme. The enzyme forms differ significantly in the size and the composition of their carbohydrate components, sensitivity towards sulfhydryl-blocking and protecting reagents, sensitivity to ferric and ferrous ions, thermostability and ability to hydrolyze some nucleotides. The more highly glycosylated form is more sensitive to thermal denaturation. AcPase I and II behave differently towards ascorbate and changes in its concentration and it is suggested that the concentration of reducing modifiers may regulate AcPase activity at the cellular level. It is hypothesized that the differing extents of glycosylation influence the structure of the enzyme forms. This is expressed in altered conformations of two enzyme forms and results in a different exposure of the essential cysteine residues.

Acid Phosphatase↗