Tryptophan analog resistance mutations in Chlamydomonas reinhardtii.

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.

Reappraisal of the genetic map of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, we have found that linkage groups XII and XIII define only a single linkage group and that linkage groups XVI and XVII also define a single linkage group. The interdigitation of the genetic maps of linkage groups XII and XIII and of XVI and XVII is presented. At present, 17 linkage groups that display Mendelian segregation have been identified in C. reinhardtii.

Complete jejunoileal necrosis due to torsion of the superior mesenteric artery.

We have reported a case of small-bowel volvulus in which complete jejunoileal necrosis resulted from torsion of the superior mesenteric artery. This case was unusual not only because of the extent of necrosis but also because primary small-bowel volvulus is rare in adults. Despite nearly total small-bowel resection, the patient continues to do well 6 months postoperatively.

Mammalian bites.

Mammalian bites account for one percent of all visits to emergency departments. More than 95% of all complications consist of local wound infections. Risk factors for infection include location on the hand or foot, puncture wounds, and treatment delay, as well as general risk factors for infection. "Clenched fist" injuries have a high risk for very severe complications. The mainstay of treatment is meticulous local wound care with debridement and irrigation. Antibiotics should only be considered as a secondary modality to prevent wound infection.

Genetic analysis of mating locus linked mutations in Chlamydomonas reinhardii.

The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt+) or the minus (mt-) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt+ locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt- locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt+/mt- diploids.

Transmission and recombination of chloroplast genes in asexual crosses of Chlamydomonas reinhardtii. II. Comparisons with observations of sexual diploids.

Patterns of biparental chloroplast gene transmission in Chlamydomonas reinhardtii diploids produced by polyethylene glycol (PEG) fusion were found to be similar to those seen in sexual diploids (VanWinkle-Swift and Birky 1978). Examination of the phenotypes of diploid clones showed that the PEG diploids produced a heterogeneous set of allelic frequencies. Neither the frequencies of parental nor recombinant phenotypes were normally distributed. In addition, frequencies of reciprocal recombinant phenotypes showed no significant correlation in the clones from individual PEG diploids. The frequencies of parental phenotypes, on the other hand, were significantly negatively correlated. Like the data from sexual diploids, this is evidence that recombinant frequencies from clones of PEG diploids are not directly related to frequencies of reciprocal recombination. The similarity of results obtained in sexual and PEG diploids supports the hypothesis that the mechanisms operating during biparental chloroplast gene inheritance in PEG diploids are identical to those in sexual diploids.

Atrazine, bromacil, and diuron resistance in chlamydomonas: a single non-mendelian genetic locus controls the structure of the thylakoid binding site.

A series of Chlamydomonas reinhardii mutants were selected for resistance to the herbicides atrazine, bromacil, and diuron. Four of these have reduced herbicide binding to the thylakoid membranes and show the non-Mendelian inheritance pattern characteristic of chloroplast genes. These mutants show a variety of selective alterations in binding of the three herbicides. These changes account for the observed patterns of in vivo cross-resistance. Analyses of chloroplast gene recombination indicate that these four mutations are in the same gene. Overall, the results suggest that this gene codes for a protein component of the herbicide binding site. One of the mutants has slow phototrophic growth and altered electron transport as has been observed in atrazine-resistant higher plant varieties, but the others are normal in these respects. The slow growth characteristic of this mutant seems to be the consequence of the same mutation which confers herbicide resistance.The mutants isolated also include a large number which achieve resistance by some secondary mechanism. These are all nuclear gene mutations, and represent numerous loci. They also show a variety of patterns of cross-resistance, but the mechanisms behind them have not yet been investigated.

Transmission and recombination of chloroplast genes in asexual crosses of Chlamydomonas reinhardii : I. Flagellar agglutination prior to fusion does not promote uniparental inheritance or affect recombinant frequencies.

Chlamydomonas reinhardii diploids homozygous for the plus mating-type (mt (+)) allele were constructed via polyethylene glycol (PEG)-induced cell fusion to investigate the transmission of chloroplast genes.We used two methods to determine whether the fusion products (PEG diploids) had inherited chloroplast markers uniparentally or biparentally. One method (multiple clone analysis) was found to markedly improve the detection of biparental transmission. With this method the frequency of biparental PEG-induced diploids was comparable to that seen in sexual diploids. Multiple clone analysis also demonstrated that fusion products which showed biparental inheritance were an extremely heterogeneous group.In some crosses, pre-treatment of one parent with isolated flagella from cells of the opposite mating type was used. This pre-treatment has been reported to promote chloroplast gene transmission from the mt (+) parent in diploids produced by PEG fusion (Adams 1982). In our crosses flagellar pretreatment had no significant effect on chloroplast gene transmission or recombination frequences. A significant bias was found for the date a cross was performed, but neither parent preferentially transmitted its alleles in all crosses. This indicates the necessity of comparing data from treated and control crosses done on the same day. We conclude from our data that separate hypotheses are not required to explain biparental inheritance in sexual versus PEG-induced diploids. The absence of biased parental allelic ratios with PEG-induced biparental diploid clones underscores their usefulness for the study of biparental gene transmission.

Non-Mendelian Inheritance of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-Resistant Thylakoid Membrane Properties in Chlamydomonas.

A uniparentally inherited 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-resistant mutant of Chlamydomonas reinhardii, Dr2, which has a resistance mechanism of the type defined as ;primary,' has been isolated. In vitro Hill reactions catalyzed by isolated thylakoid membranes reveal a reduced apparent affinity of the thylakoids for DCMU. These changes in membrane properties quantitatively account for the resistance of mutant Dr2 to herbicide inhibition of growth. The properties of this mutant show that all of the Hill reaction-inhibiting DCMU binding sites are under identical genetic control. Mutant Dr2 is a useful new uniparental genetic marker, since it has a novel phenotype and it may be possible to identify its altered gene product. The low cross-resistance of Dr2 to atrazine suggests that there may be considerable flexibility in exploiting induced herbicide resistance of crop plants for improving herbicide specificity.Four mendelian mutants in at least three loci all have resistance mechanisms in the class we define as ;secondary.' They are as sensitive as wild type to in vitro inhibition of the Hill reaction, and must acquire resistance in vivo by preventing the active form of the herbicide from reaching the sensitive site.

Activation of protein mediators of inflammation and evidence for endotoxemia in Borrelia recurrentis infection.

Fifteen patients with Borrelia recurrentis infection were studied to evaluate the role of certain plasma proteins and endotoxin in the pathophysiology of both the acute illness and the Jarisch-Herxheimer-like reaction. The causative spirochetes disappeared from the blood during the Jarisch-Herxheimer-like reaction, which occurred about 2 hours after antibiotic therapy. The mean titers of Hageman factor, plasma prekallikrein and serum hemolytic complement activity were decreased at the time of admission and 2 hours after treatment, and rose to normal values during convalescence. Serum properdin titers were decreased in 14 patients at the time of admission, in 12 patients 2 hours after treatment, and in none during convalescence. The frequency of elevated levels of fibrinogen-related antigens increased from three patients at the time of admission to 12 patients 2 hours after treatment. Results of plasma limulus tests for endotoxin-like material were positive in 11 patients at the time of admission and in 13 patients 2 hours after treatment. These findings demonstrated that Hageman factor, prekallikrein and proteins of the complement system are activated in B. recurrentis infection and that endotoxin may play a role in both the acute illness and in the development of the Jarisch-Herxheimer-like reaction after treatment.