Growth In Complex Medium Research Articles

Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression.

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis.

Effects of elevated temperature on bacterial community structure and function in bioreactors treating a synthetic wastewater

The impact of elevated temperature on bacterial community structure and function during aerobic biological wastewater treatment was investigated. Continuous cultures, fed a complex growth medium containing gelatin and α-lactose as the principal carbon and energy sources, supported mixed bacterial consortia at temperatures ranging from 25–65°C. These temperature- and substrate-acclimated organisms were then used as inocula for batch growth experiments in which the kinetics of microbial growth and substrate utilization, efficiency of substrate removal, and mechanism of substrate removal were compared as functions of temperature. Bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) revealed that distinct bacterial consortia were supported at each temperature. The efficiency of substrate removal declined at elevated temperatures. Maximum specific growth rates and the growth yield increased with temperature from 25–45°C, but then decreased with further elevations in temperature. Thus, maximum specific substrate utilization rates did not vary significantly over the 40°C temperature range (0.64 ± 0.04 mg COD mg−1 dry cell mass h−1). A comparison of the degradation of the protein and carbohydrate portions of the feed medium revealed a lag in α-lactose uptake at 55°C, whereas both components were utilized simultaneously at 25°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 140–145.

Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker.

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.

Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow

Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy. Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH. In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium. Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1. When C. cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition. In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited. These data indicate that C. cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose. This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C. cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing.

Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture.

We have previously shown that fetal lung mesenchyme can reprogram embryonic rat tracheal epithelium to express a distal lung phenotype. We have also demonstrated that embryonic rat lung epithelium can be induced to proliferate and differentiate in the absence of lung mesenchyme. In the present study we used a complex growth medium to induce proliferation and distal lung epithelial differentiation in embryonic tracheal epithelium. Day-13 embryonic rat tracheal epithelium was separated from its mesenchyme, enrobed in growth factor-reduced Matrigel, and cultured for up to 7 days in medium containing charcoal-stripped serum, insulin, epidermal growth factor, hepatocyte growth factor, cholera toxin, fibroblast growth factor 1 (FGF1), and keratinocyte growth factor (FGF7). The tracheal epithelial cells proliferated extensively in this medium, forming lobulated structures within the extracellular matrix. Many of the cells differentiated to express a type II epithelial cell phenotype, as evidenced by expression of SP-C and osmiophilic lamellar bodies. Deletion studies showed that serum, insulin, cholera toxin, and FGF7 were necessary for maximum growth. While no single deletion abrogated expression of SP-C, deleting both FGF7 and FGF1 inhibited growth and prevented SP-C expression. FGF7 or FGF1 as single additions to the medium, however, were unable to induce SP-C expression, which required the additional presence of serum or cholera toxin. FGF10, which binds the same receptor as FGF7, did not support transdifferentiation when used in place of FGF7. These data indicate that FGF7 is necessary, but not sufficient by itself, to induce the distal rat lung epithelial phenotype, and that FGF7 and FGF10 play distinct roles in lung development.

Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis.

Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.

MOLECULAR IDENTIFICATION AND CHARACTERIZATION OF AVIAN MYCOPLASMAS

Serological techniques are recommended procedures for the identification of Mycoplasma species. They enable separation of mycoplasmas at the species level and are widely used for practical laboratory identification of the isolated microorganisms. However, complex growth media are required for the isolation of Mycoplasma species and some strains of pathogenic Mycoplasma species grow very fastidious and can not be cultured. Introduction of DNA based diagnostic methods in mycoplasma research enabled survey of the whole genome in a single assay and reveal also information about non-coding regions of the genome. Restriction enzyme analysis is a powerful method for differentiation of close related strains but requires high quality of DNA which can only be obtained from cultured mycoplasmas. The increasing number of cloned mycoplasma genomic fragments are successfully used for identification and differentiation of mycoplasma strains and species in hybridization studies. The polymerase chain reaction enables identification and characterization of mycoplasmas without culture directly from clinical material. Large genomic sequencing projects produced complete nucleotide sequence from two Mycoplasma species and several more are in progress. This data will open the possibility to understand better the biology of mycoplasmas.

Molecular mode of action of the antifungal beta-amino acid BAY 10-8888.

BAY 10-8888 is a cyclic beta-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H+-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a KT (Michaelis constant of the transport reaction) of 0.95 mM and a Vmax of 18.9 nmol x min-1 x 10(7) cells-1. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.

Phenotypic variation and survival of genetically marked Pseudomonas tolaasii in mushroom compost

The role of Pseudomonas tolaasii as an important pathogen of the common mushroom Agaricus bisporus is difficult to study in the microbially complex growth medium used for mushroom production. Two strains of P. tolaasii that had been marked with kanamycin resistance and xylE genes were introduced individually into casing soil over mushroom compost. Survival studies revealed that P. tolaasii numbers in casing soil over mushroom compost decreased a 1000-fold in the first 9 days and then remained relatively stable over the rest of the monitoring period. The presence of the pathogenic colony form and the nonpathogenic colony variant was monitored on mushroom caps and in mushroom compost to detect any phenotypic variation while incubated in these environments. Reversion from the nonpathogenic to pathogenic form was detected following isolation and culture from diseased mushroom caps. Inoculation of the marked strains directly onto the cap or into compost beds seeded with A. bisporus resulted in the appearance of brown blotch symptoms.Key words: Pseudomonas tolaasii, Agaricus bisporus, phenotypic variation, mushroom disease.

Quality-control testing of mycoplasma medium.

Mycoplasmas lack many of the biosynthetic pathways of higher organisms; consequently, their growth and survival depend on an external supply of a wide variety of nutrients. Because they are very fastidious in their nutritional requirements, a complex growth medium is required for their culture. Although the exact requirements of most mycoplasma species are still incompletely defined, most can be grown in the laboratory in both liquid and solid agar media formulated to comprise as a minimum: a broth base, yeast extract, serum source, and solutions of pure chemicals. Details of components added to any particular medium will vary, according to the species of mycoplasma it is intended to culture (see Chapters 4– 6). It is clear that such a large number of components must inevitably introduce a degree of variability to the growth-promoting properties of the mycoplasma media between formulation lots. Consequently, it is of paramount importance that the introduction of any new component into a formulation and the preparation of media lots should always be accompanied by a quality validation with mycoplasma strains that the particular formulation is intended to support. This quality-control (QC) testing should cover all components, including preparations of pure chemicals (e.g., glucose and arginine) because although intrinsically these chemicals are unlikely to vary between lots supplied by the same manufacturer in the same way that broth base, serum, and yeast extract might, it is still necessary to exclude the possibility that errors may have occurred during their preparation (see Note 1).

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media.

Extracellular production of a hybrid beta-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors.

Cultivation conditions for the extracellular production of a hybrid beta-glucanase from Bacillus were established by using Escherichia coli JM 109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid beta-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of beta-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of beta-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of beta-glucanase [E. coli JM 109(pLF3)] was tested on the fermenter scale.

Nutrient utilization and transport in the thermoacidophilic archaeon Sulfolobus shibatae

Growth of the thermoacidophilic archaeon Sulfolobus shibatae was tested on a range of carbon and nitrogen sources. Optimal defined and complex growth media were developed and growth conditions in both shake flask and fermenter cultures were optimized. Better growth was observed on maltose in particular and disaccharides in general than on monosaccharides. Moreover, maltose utilization was not repressed in the presence of glucose which suggests that glucose is not the preferred substrate of S. shibatae. Uptake studies putatively identified two saturable, constitutive maltose transport systems, a high-affinity, possible membrane-binding system with a K m of 20 μM and a V max of 218 nmol min−1 (mg protein)−1, and a low-affinity, proton-dependent system with a K m of 158 μM and a V max of 680 nmol min−1(mg protein)−1. Both systems showed differential responses to treatment with 2,4-dinitrophenol and arsenate, and differed from other maltose transport mechanisms described in being constitutive under all conditions tested and not repressed by glucose.

X-ray photoelectron spectroscopy analysis of the surface composition of Azospirillum brasilense in relation to growth conditions

X-ray photoelectron spectroscopy (XPS) has been used to determine the surface chemical composition of the Gramnegative bacterium Azospirillum brasilense as a function of growth conditions. As opposed to growth in complex medium, growth in synthetic medium led to low reproducibility of XPS results, and did not show a significant variation of the cell surface composition as a function of culture time; moreover, the molecular composition obtained by modelling the XPS data was found to be generally poorer in proteins, and richer in polysaccharides and in hydrocarbon-like compounds. The direct correlation between the phosphate concentration and the concentration of carbon bound only to carbon and hydrogen, C (C,H), indicated that hydrocarbon-like compounds detected at the surface of cells grown in synthetic medium are essentially phospholipids. The poor reproducibility of the XPS data obtained for cells grown in synthetic medium and the random observation of phospholipids at the outermost cell surface are not due to cell disruption or to migration of intracellular components during sample preparation; exposure of phospholipids at the cell surface reflects either actual variations of the composition of the native surface induced by nutrient limitation or reorganization of cell surface polymers upon freeze drying.

Purification and Characterization of an Extracellular Aspartate Protease fromPhycomyces blakesleeanus

De Vicente, J. I., De Arriaga, D., Del Valle, P., Soler, J., and Eslava, A. P. 1996. Purification and Characterization of an Extracellular Aspartate Protease fromPhycomyces blakesleeanus. Fungal Genetics and Biology20,115–124. An acid protease has been found in the culture broth ofPhycomyces blakesleeanusgrowing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease ofPhycomycesmigrated as a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A–Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70°C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50°C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-NO2)-Arg-Leu as the substrate, theKm,kcat, andVmaxvalues were 8.78 μM, 1.25 s−1, and 2.12 μmol min−1mg−1, respectively. The protease was insensitive to phenylmethylsulfonyl fluoride,O-phenanthroline,N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with aKivalue of 1.33 nM.The data suggest that this protease has properties of an aspartate-type proteinase.

Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens

Amino acid consumption by Entamoeba histolytica and E. invadens has been measured in order to assess the possible roles of amino acids as energy substrates. Mixtures of amino acids enhanced the growth of the parasites in complex medium and their survival in simple medium. The consumption of several amino acids by the parasites suspended in simple media was greater when glucose was absent, suggesting that they may act as alternative energy sources. Under these conditions, asparagine was consumed extremely rapidly by E. histolytica in particular, and arginine, leucine and threonine were used greatly by both species. There was also a marked consumption of aspartate, but this occurred even when glucose was present. These five amino acids and phenylalanine were the ones consumed in greatest amounts during growth of E. histolytica in complex medium. Under the same growth conditions, E. invadens also used asparagine, arginine, leucine and threonine and in addition there was a large consumption of serine and especially glutamate. In contrast, the aspartate concentration in the complex medium increased and there was also a net increase in the concentration of some other amino acids. Alanine was produced by both species when the parasites were incubated in simple medium with glucose, and in greater amounts during growth in complex media, suggesting that it is an end product of energy metabolism. The findings provide support for the suggestion that energy generation through amino acid catabolism may be a characteristic feature of anaerobic parasitic protists.

Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens.

Amino acid consumption by Entamoeba histolytica and E. invadens has been measured in order to assess the possible roles of amino acids as energy substrates. Mixtures of amino acids enhanced the growth of the parasites in complex medium and their survival in simple medium. The consumption of several amino acids by the parasites suspended in simple media was greater when glucose was absent, suggesting that they may act as alternative energy sources. Under these conditions, asparagine was consumed extremely rapidly by E. histolytica in particular, and arginine, leucine and threonine were used greatly by both species. There was also a marked consumption of aspartate, but this occurred even when glucose was present. These five amino acids and phenylalanine were the ones consumed in greatest amounts during growth of E. histolytica in complex medium. Under the same growth conditions, E. invadens also used asparagine, arginine, leucine and threonine and in addition there was a large consumption of serine and especially glutamate. In contrast, the aspartate concentration in the complex medium increased and there was also a net increase in the concentration of some other amino acids. Alanine was produced by both species when the parasites were incubated in simple medium with glucose, and in greater amounts during growth in complex media, suggesting that it is an end product of energy metabolism. The findings provide support for the suggestion that energy generation through amino acid catabolism may be a characteristic feature of anaerobic parasitic protists.

The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity

The growth of Listeria monocytogenes ATCC 23074 in defined medium is sensitive to high osmolarity when compared with its growth in complex media, such as brain heart infusion (BHI). The two major contributors to this difference in growth rate are the availability in BHI of the osmoprotectant glycine betaine and peptides. Peptone plays two major roles: firstly as a nutritional supplement for protein synthesis, and secondly as a source of amino acids and peptides that serve as a mechanism of maintaining turgor. In the presence of peptone the total amino acid pool at high osmolarity is substantial and even in the presence of glycine betaine the amino acid pool makes a major contribution to turgor maintenance. At high osmolarity there is a general increase in amino acid pools, with particularly substantial pools of glutamate, aspartate, proline, hydroxyproline and glycine. Peptides are also accumulated by cells from the peptone supplied in the medium. Glycine-containing peptides are accumulated in the cytoplasm under all conditions. Specific glycine- and proline-containing peptides stimulate growth at high osmolarity. The peptide prolyl-hydroxyproline accumulates in cells to high levels in response to growth at high osmolarity, and the pools of the derived amino acids also show a dependence on the external osmotic pressure. However, proline only confers significant osmoprotection when supplied as peptides. The significance of these data in the context of the occurrence of L. monocytogenes in foods with high peptide content is discussed.

10] Tumor cell culture

Publisher Summary This chapter describes generally successful methods for culture initiation of tumor cells from multiple human and animal source tissues. The success rate of 80-90% for the initiation of viable primary cultures with the propensity to maintain the ability to divide and differentiate for at least two to three subcultures (which is 4-10 population doublings, depending on the culture) has been observed for the attempted culture of more than 10,000 source specimens from surgically resected and fetal donor tissues. The greatest chance for successful cell line establishment is from metastatic tumors or those with high metastatic potential, particularly tumors of blood cell origin, melanomas, and solid tumors that have the ability to grow in suspension as effusions or ascites. Most of the tumor cells need numerous growth factors and nutrients for in vitro growth including (1) Low seeding densities are often lethal to tumor cell cultures, particularly those derived from primary tumors. (2) Metastatic tumors, especially those in pleural effusions or ascites, are generally easier to grow and can be propagated in less complex growth media. (3) There is a general inability to readily develop cell lines using standard limiting dilution or semisolid media cloning methods.

Ammonium-mediated reduction of plasmid copy number and recombinant gene expression in Escherichia coli.

The effect of ammonium as a medium supplement on plasmid-encoded recombinant beta-galactosidase synthesis was explored in Escherichia coli cells during aerobic growth in complex medium. After induction, only doses of ammonium chloride below 1 g/L are able to transiently enhance the yield. However, the presence of nontoxic ammonium chloride concentrations of up to 10 g/L results in lower values of beta-galactosidase in a concentration-dependent fashion. A significant reduction in plasmid DNA content explains the decrease in the yield by a gene-dosage-involving mechanism.