Genetic and developmental characteristics of a new color variant, axanthic, in the Mexican axolotl, Ambystoma mexicanum Shaw

A new genetic trait, axanthic, is described in the Mexican axolotl with a phenotype completely lacking visible xanthophores and iridophores. Animals received from the colony of Dr. L. E. DeLanney at Ithaca, New York, have been mated among themselves for a total of seven spawnings, in which the axanthic trait was inherited as a simple Mendelian recessive. The symbol for this newly found gene isax. Homozygous recessive animals appear normal except for their lack of visible xanthophores and iridophores. The results of the spawnings also indicate that the axanthic gene is not linked to the melanoid gene (m). Its linkage with other known mutants in the axolotl is being tested.     

The developmental genetics of pigment mutants in the Mexican axolotl

The Mexican axolotl (Ambystoma mexicanum) provides a well-defined set of color genes which are useful for various types of analyses. These include the a (albino), m (melanoid), ax (axanthic), and d (white) genes. In addition, various combinations of these genes and a number of as yet undescribed mutants also exist. Three of these mutants (a, ax, and m) have defects associated with specific neural-crest-derived pigment cell types. The fourth mutant (d) appears to provide an unsuitable environment for the migration and maintenance of pigment cells. In one case (m), detailed information concerning the specific nature of the genetic defect is available. The goal of this article is to demonstrate ways in which the existing information on the axolotl color genes can best be utilized in terms of understanding not only the mutant phenotypes, but basic concepts in the cell and developmental biology of pigmentation as well. Thus, an attempt has been made to sort through the genetic and biochemical data relevant to these mutants in order to stimulate renewed interest in a more detailed pursuit of such studies.     

Mitogen-induced B-cell differentiation in Xenopus laevis

Abstract. Four genes are known to affect pigmentation in the Mexican axolotl. The purpose of this article is to review previous information pertinent to these genes and to reevaluate such information in light of new evidence that demonstrates (in a preliminary way) how pigments, and subsequently phenotypes, are affected by the various pigment genes. Each of the mutant phenotypes – m (melanoid), ax (axanthic), a (albino), and d (white) - is compared to the wild type (D). All of these genes are recessives, all of them affect phenotypic changes during development, and three of the four ( m, a , and d ) also affect specific biochemical (i.e., pigment) changes during development. In the axolotl, color patterns can be directly correlated to the presence(or absence) of a variety of pigments that are normally found in discrete pigment cells. Qualitative and quantitative analyses of the bright-colored pigments (pteridines and flavins in this case) present in axolotl skin demonstrate that these pigments vary significantly among the various phenotypes under consideration. These analyses raise some interesting questions with regard to how each of the pigment genes is believed to act, and numerous possibilities for continued experimentation are suggested.     

Pigment cell differentiation: the relationship between pterin content, allopurinol treatment, and the melanoid gene in axolotls

The effects of allopurinol (an inhibitor of the enzyme xanthine dehydrogenase (XDH] and the melanoid gene on pigment cell differentiation in the axolotl were examined by analyzing pigment components of the xanthophore (pterins). Pterin contents of skin extracts (70% ethanol) from wild type, allopurinol-treated and melanoid axolotls were determined by thin layer chromatography (TLC) and fluorometric scanning of TLC plates. Heights of peaks produced were used as a quantitative measure for pterin content. Results reveal that melanoid animals contain significantly reduced amounts of all seven pterins examined as compared with wild type animals. Allopurinol-treated animals have reduced levels of four pterins (xanthopterin, isoxanthopterin, biopterin and sepiapterin) as compared with the wild type. These findings suggest that the alterations in pterin biosynthetic pathways, either by drug-induced inhibition of XDH activity or by the melanoid gene, produce similar dramatic changes in pigment phenotype which are manifested by alterations in pigment cell differentiation.     

Dosage effects of the white (d) and melanoid (m) genes on pigment pattern in the Mexican axolotl,Ambystoma mexicanum,Shaw

Two unlinked autosomal recessive genes, white (d) and melanoid (m), are known to affect wild-type coloration in the axolotl. The d allele in the homozygous recessive condition reduces the total number of melanophores and restricts their migration to the top of the head and along the spinal column. The m allele in the homozygous recessive condition increases the number of melanophores and effects a more uniform distribution. The action of these genes was further investigated by studying the phenotypes of triploid larvae of known genotype at the d and m loci. Triploidy was induced by heat treating the eggs immediately after oviposition to inhibit the second maturation division. Triploidy was verified by chromosomal counts in epidermal cells of tail-tip preparations. Melanophore distribution and number were recorded at 16 and 30 days after oviposition. Two d alleles in Ddd triploids effect a reduction in melanophore number and their rate of proliferation, but have no effect on melanophore distribution. Two m alleles in Mmm triploids effect an increase in the number of melanophores and their rate of proliferation, but have no effect on their distribution. Therefore, it appears that there are at least two separate physiological actions of gene D and M, one that determines melanophore distribution and another that determines their number and rate of proliferation.     

Allopurinol-induced melanism in the tiger salamander (Ambystoma tigrinum nebulosum).

The enzyme, xanthine dehydrogenase (XDH), has been examined in Ambystoma tigrinum nebulosum with respect to its role in pigmentation. It now seems probable that the melanoid gene (m) either codes directly for XDH or is somehow intimately connected with the normal function of this enzyme. Inhibition of XDH using the drug, allopurinol, results in animals which appear to be phenocopies of melanoid mutants as described for the Mexican axolotl (Ambystoma mexicanum). The effects of allopurinol in terms of specific pigmentary alterations were examined, and a new method for analyzing heterogeneous extracts of skin pigments (e.g., purines and pteridines) is presented. The significance of the link between XDH and melanism is discussed with emphasis on possible mechanisms of pigment induction and general applicability to biological systems.     

A regulatory interaction between pyrimidine and purine biosyntheses via ureidosuccinic acid

Summary l-Ureidosuccinic acid (USA), an intermediate metabolite of pyrimidine biosynthesis, inhibits cell growth. Mutants blocked in pyrimidine biosynthesis after dihydroorotate (DHO), are more resistant to USA than are the wild type and the mutants blocked in the synthesis of DHO. This difference in sensitivity correlates with the level of dihydroorotase activity. Purines revert USA inhibition. Mutants selected for their resistance to USA were resistant because of a) overproducing purines (adenine excretion), b) having a reduced USA uptake, or c) showing a USA resistance independant of the above phenotypes.     

On the Development of Pigment Patterns in Amphibians

SYNOPSIS. Pigmentary mutants in amphibians provide importantvehicles for studying basic problems in development. Some ofthese mutants exert influences on the tissue environment inwhich the chromatophores differentiate and others involve theexpression of pigment in specific types of pigment cells. Melanophoresare the best known of all chromatophores, and albinism has beenmuch studied. Genes controlling its expression may operate atdifferent levels. Some are involved in the production of tyrosinase,whileothers affect the melanosomal matrix. In contrast, melanoidmutants are characterized by an overproduction of eumelanin,usually through the differentiation of an excessive number ofmelanophores. Melanoid mutants of the axolotl also exhibit a great diminutionin xanthophore and iridophore number, and the same seems tobe true of some melanoid-like mutants of leopard frogs. Thepteridine pigments of xanthophores and purines of iridophoresare often products of xanthine dehydrogenase (XDH) activity,and it is suspected that the expression of the melanoid phenotypemay result from a genetic defect involving this enzyme. Thisis supported by experiments involving the administration ofan XDH inhibitor, allopurinol, to normal larvae. This inhibitorresults in the production of partial phenocopies of the melanoidcondition. Blue mutants involving either partial or completediminution of xanthophore and iridophore pigments may also bebased upon deficient XDH activity.     

Superoxide Dismutase Activity in Pseudomonas putida Affects Utilization of Sugars and Growth on Root Surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

The ntrB and ntrC Genes Are Involved in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

Azospirillum brasilense Sp7 and its ntrA (rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the ntrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC and ntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and ntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that the ntrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilense Sp7.     

Inactivation of genes,encoding tocopherol biosynthetic pathway enzymes,results in oxidative stress in outdoor grown Arabidopsis thaliana

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes – tocopherol cyclase (vte1) and γ-tocopherol methyltransferase (vte4) – were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components.     

Characterization of the ntrBC genes of Azospirillam brasilense Sp7: Their involvement in the regulation of nitrogenase synthesis and activity

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif⁺. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.     

Use of the Mud(Ap,lac) bacteriophage to study the regulation of L-proline biosynthetic genes inEscherichia coli K-12

One operon fusion to the promoter of either theproA orproB genes of the proline biosynthetic pathway was obtained by the use of the Mud(Ap,lac) bacteriophage. This operon fusion was further stabilized by transformation with the plasmid pGW600 containing the wild type Mu repressor gene. The level of β-galactosidase in this strain was not affected by the presence of high concentrations of NaCl in the growth medium. Mutations affecting the regulation of thispro-lac genetic fusion were generated by the insertion of Tn5; β-galactosidase levels in these mutants were higher than in the parental strain when proline was present at a high level. In some of these mutants we observed either repression or maintenance of β-galactosidase levels whenpro-lac (F′proAB ⁺) merodiploids were constructed.     

Characterization of the Staphylococcus aureus chromosomal gene pcrA,identified by mutations affecting plasmid pT181 replication

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapBl), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds. scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes apear to be inducible by homocysteine and repressible by methionine (or Sadenosylmethionine).