The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: Homozygous strains

The specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time. The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.     

Heterozygous diploid strains of Aspergillus nidulans: Enhanced virulence for mice in comparison to a prototrophic haploid strain

The paper describes quantitative studies of the virulence for DBA/2J mice of three heterozygous diploid strains ofAspergillus nidulans synthesized from avirulent auxotrophic haploid strains, and auxotrophic strains of reduced virulence. These studies demonstrate that the heterozygous diploid strains possess an unusually high virulence for mice in comparison to a haploid prototrophic strain ofA. nidulans employed as a virulence standard.     

A further study on chromosome minimization by protoplast fusion in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1–12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 μg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains

It was shown previously that the specific catalase activity of human diploid cell strains falls immediately after subculture and then progressively rises in an exponential fashion. In this paper evidence is presented suggesting that the rise in catalase activity cannot be due to an accumulation within the cell of a small molecule which enhances enzyme activity in cell-free extracts. It is also shown that activity per cell, as well as per unit cell protein, rises as the culture grows. The rate of fall of specific catalase activity immediately after subculture is greater if the cells are at a low population density than if they are at a high one. The rate of fall can be made more sharp by increasing the frequency with which the cultures are fed. It is shown that used medium, which has previously been incubated with cultured cells of the same strain, does not significantly change either the rate of fall of specific catalase activity following subculture, or the rate of its subsequent rise. It is postulated, as one possibility, that the cells liberate into the medium an enhancer of cell catalase activity which is highly labile. The steady state concentration of this enhancer in the medium might be expected to increase as the culture grew, but to decrease when the cells are subcultured into fresh medium or when the frequency of feedings is increased.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Studies on the regulation of the three enzymes of the leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, α-D-galactose-1-phosphate:UDP glucose uridylyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increased cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.     

Ribosomal DNA magnification in Saccharomyces cerevisiae.

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.     

Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

Insight into the basis of root growth in Arabidopsis thaliana provided by a simple mathematical model

Plant organ growth changes under genetic and environmental influences can be observed as altered cell proliferation and volume growth. The two aspects are mutually dependent and intricately related. For comprehensive growth analysis, it is necessary to specify the relationship quantitatively. Here, we develop a simple mathematical model for this purpose. Our model assumes that the biological activity of a given organ is proportional to the cell number of the organ and is allocated into three aspects: cell proliferation, volume growth, and organ maintenance. We analyzed the growth of primary roots of Arabidopsis thaliana (L.) Heynh. in one tetraploid and four diploid strains using this model. The analysis determined various growth parameters, such as specific cost coefficients of cell proliferation and volume growth for each strain. The results provide insight into the basis of interstrain variations and ploidy effects in root growth.     

Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea-pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC., was exposed to increasing concentrations of INH. Two INH-resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH-sensitive strain was virulent for guinea-pigs, the INH-resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.     

Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity

Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.     

The structural genes of internal invertases in Saccharomyces cerevisiae.

Summary Polyacrylamide gel electrophoresis (without SDS) of invertases from strains each carrying only one of the five known SUC-genes revealed differences in mobility of the internal enzymes. SUC1 invertase moved distinctly slower than the invertases formed in the presence of genes SUC2 to SUC5. Three bands of internal invertase activity were found in diploids carrying both SUC1 (slow invertase) and one of the other SUC-genes (fast invertases). Tetrad analysis of such diploids yielded haploids which showed the same three bands if they carried SUC1 in combination with another SUC gene. A gene dosage effect was observed in relation to invertase activity in haploid strains with only gene SUC1 or only SUC4 on one hand, and both genes on the other hand. A sucrose non-fermenting and invertase negative strain with mutant allele suc3-3 of gene SUC3 (fast invertase) was crossed with SUC1. The heterozygous diploid and the recombinant haploids (SUC1 suc3-3) showed two bands in the region of the internal invertase: a slow SUC1 band and a second band corresponding to the intermediate band of SUC1-SUC3 strains. The intermediate band in SUC1 suc3-3 strains is considered as a hybrid consisting of an active SUC1-monomer and an inactive suc3-mutant monomer. Formation of such hybrid bands was taken as evidence for the structural nature of SUC-genes.     

myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth

We have identified and cloned a novel essential myosin I in Aspergillus nidulans called myoA. The 1,249-amino acid predicted polypeptide encoded by myoA is most similar to the amoeboid myosins I. Using affinity-purified antibodies against the unique myosin I carboxyl terminus, we have determined that MYOA is enriched at growing hyphal tips. Disruption of myoA by homologous recombination resulted in a diploid strain heterozygous for the myoA gene disruption. We can recover haploids with an intact myoA gene from these strains, but never haploids that are myoA disrupted. These data indicated that myoA encodes an essential myosin I, and this has allowed us to use a unique approach to studying myosin I function. We have developed conditionally null myoA strains in which myoA expression is regulated by the alcA alcohol dehydrogenase promoter. A conditionally lethal strain germinated on inducing medium grows as wild type, displaying polarized growth by apical extension. However, growth of the same myoA mutant strain on repressing medium results in enlarged cells incapable of hyphal extension, and these cells eventually die. Under repressing conditions, this strain also displays reduced levels of secreted acid phosphatase. The mutant phenotype indicates that myoA plays a critical role in polarized growth and secretion.     

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. II. A search for quantitative interrelationships between the three enzyme activities.

Studies on a normal human diploid cell strain revealed that the specific activity of the cell protein, for each of the three enzymes of the Leloir pathway, changed significantly as the cells grew. The kinetics of change in specific activity varied according to the enzyme being studied, and the kinetics for each enzyme varied from experiment to experiment. Within each experiment, there was no consistent correlation between specific activity for any one enzyme and specific activity for the other two. The ratios between the specific activities did not tend to remain constant as the absolute levels of specific activity changed. Hence, the activities did not behave coordinately. The kinetics of change in these ratios varied from experiment to experiment. The failure of galactose to stimulate increased cellular activity for the three enzymes (shown in the preceding paper), and the absence of a coordinate relationship between the activities, represent a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL.Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size.The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1xpab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned.These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.     

Studies on symbiotic nitrogen fixation by a new strain of tetraploid red clover (UO36)

Summary Some characteristics of the symbiotic nitrogen fixation by tetraploid red clover (UO36) have been studied in laboratory experiments. Cross-inoculations were made on tetraploid and diploid clover plants usingRhizobium isolates from both types of clover. Nodulation activity and nitrogen fixation were determined. No sign of specific strains necessary for optimal fixation of nitrogen by theUO36-symbiosis was observed in comparison to the diploid clover strain.Rhizobium cultures effective in connection with cultivation of diploid clover will probably also be effective when cultivating tetraploid clover strains.     

Chromosomal changes in eight cell strains of the tasmanian rat-kangaroo,Potorous tridactylis (Marsupialia). Male and female heart fibroblasts cultured in vitro

Eight cell strains, derived from the hearts of a male and a female Motorous, were followed during their in vitro cultivation.All three male cell strains started as normal diploid cell strains. One of them, 2Hpo stayed diploid until passage 59, when the cells were frozen and stored at –96°C. After a period of growth retardation, that lasted two months, 1Hpo showed aneuploidy, the cells having 22–24 chromosomes. The cells with 23 chromosomes formed about 30% of the population. These cells predominantly missed the chromosomes 2, 3 and Y₁, from the tetraploid set. In the other cells no consistent pattern was observed. The cell strain 4Hpo did not show aneuploidy after three months of growth retardation. At the last passage (nr. 24) before death, it showed 25% diploid cells and 40% tetraploid cells.Three female strains were initiated on fibrin clot, two on plasm clot. No differences in growth and chromosomal changes, due to the different embedding media, were observed. All the strains started as diploid (2n=12) cell strains. The chromosomal changes that occurred showed many differences. Three cell strains (5Hf, 7Hp, 52Hf) died without showing any pattern in the aneuploid cells. One cell strain (53Hf) showed an aneuploid cell population with a stemline of 14 chromosomes. The cell strain (8Hp) showed different changes in ploidy. After 50 passages, it changed from diploid to aneuploid (19 chromosomes per cell in the stemline). Twenty passages later diploid cells started to dominate the population again (80% at passage 85). Then a new aneuploid population with a stemline of 18 chromosomes (30% triploid cells) arose, and the strain survived as a permanent line.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract Nr. 052-64-I BIAN, and it also received support form the Foundation for Basic Medical Research (FUNGO).     

Novel genetic components controlling invertase production in Saccharomyces cerevisiae

Low levels of invertase (EC 3.2.1.26) activity were observed in most diploid strains of S. cerevisiae used in this work. There was no effect of mating type on invertase levels, and cell surface was not a limiting factor, because an increase in ploidy did not cause further decrease in specific invertase activity. Finally, some diploids showed the activity expected from the additive effects of different SUC genes, and haploid strains possessing two SUC genes expressed very variable invertase activities depending on the strain. This suggested the existence of one or more additional genes which control the levels of invertase. Genetic analysis of SUC5 strains provided evidence of the existence of a new gene, RPS5, which drastically reduced the specific invertase activity in strains possessing active SUC alleles. The recessive allele of this gene (rps5) allows expression of higher levels of invertase. We suggest that genes similar RPS5 are responsible for the low levels of invertase activity observed in diploid strains of S. cerevisiae.     

Industrial Application of Artificially Induced Diploid Strains of Torulaspora delbrueckii

Diploid strains of Torulaspora delbrueckii were tested for industrial application. Because the cell volume of the diploid strain was three times as large as that of the parental haploid strain, collection and subsequent dehydration to make compressed yeast cakes were greatly improved with the diploid YL3. The time required for dehydration of the diploid strain was shortened to 1/2.5 that of the parent strain under conventional conditions. Moreover, for the diploid cells frequent filter changes for dehydration were not required, which was the case with parental cells. Fermentation activity and tolerance to freeze-thawing in dough were succesfully inherited by the diploid strains. The diploid YL3 showed nearly the same activity as the diploid F31 in bread making. However, the endurance period of yeast cakes when stored at 30°C without softening to lead to liquefaction was much longer in YL3 (199 h) than in F31 (132 h). This superiority was ascribed to the fact that YL3 was induced through direct diploidization and had no genetic defect on chromosomes because the wild-type strain was employed as the parent, whereas F31 was obtained through protoplast fusion from two auxotrophic mutants and carried at least two mutagenized genes that were masked by heterolallelism.     

Hydrogen Peroxide Resistance of Acetobacter pasteurianus NBRC3283 and Its Relationship to Acetic Acid Fermentation

The bacterium Acetobacter pasteurianus can ferment acetic acid, a process that proceeds at the risk of oxidative stress. To understand the stress response, we investigated catalase and OxyR in A. pasteurianus NBRC3283. This strain expresses only a KatE homolog as catalase, which is monofunctional and growth dependent. Disruption of the oxyR gene increased KatE activity, but both the katE and oxyR mutant strains showed greater sensitivity to hydrogen peroxide as compared to the parental strain. These mutant strains showed growth similar to the parental strain in the ethanol oxidizing phase, but their growth was delayed when cultured in the presence of acetic acid and of glycerol and during the acetic acid peroxidation phase. The results suggest that A. pasteurianus cells show different oxidative stress responses between the metabolism via the membrane oxidizing pathway and that via the general aerobic pathway during acetic acid fermentation.