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Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange.

Helicobacter pylori is one of the most common human pathogens. It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma. Helicobacter mustelae is a bacterium closely related to H. pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections. Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors. The flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the major flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin genes from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequence of the H. pylori gene proved that it encoded the second flagellin molecule found in H. pylori flagellar filaments. The genes were named flaB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flagellin was much lower (58%). Both flaB genes were preceded by sigma 54-like promoter sequences. Mapping of the transcription start site for the H. pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing. Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule. Whereas flaA mutants were completely nonmotile, flaB mutants retained motility.

Amino Acid Sequence↗

Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster.

Both Proboscipedia (PB) and Sex Combs Reduced (SCR) activities are required for determination of proboscis identity. Here we show that simultaneous removal of PB and SCR activity results in a proboscis-to-antenna transformation. Dominant negative PB molecules inhibit the activity of SCR indicating that PB and SCR interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of PB and SCR and the ability of PB and SCR to interact in a multimeric complex control the determination of four adult structures. The absence of PB and SCR expression leads to antennal identity; expression of only PB leads to maxillary palp identity; expression of only SCR leads to tarsus identity; and expression of both PB and SCR, which results in the formation of a PB-SCR-containing complex, leads to proboscis identity. However, the PB-SCR interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the PB-SCR interaction may be regulated and indirect. This regulation may also explain why ectopic expression of SCR(Q50K) and SCR do not result in the expected transformation of the maxillary palp to an antennae and proboscis, respectively. Previous analysis of the requirements of SCR activity for adult pattern formation has shown that ectopic expression of SCR results in an antenna-to-tarsus transformation, but removal of SCR activity in a clone of cells does not result in a tarsus-to-arista transformation. Here we show in five independent assays the reason for this apparent contradictory requirement of SCR activity in tarsus determination. SCR activity is required cell nonautonomously for tarsus determination. Specifically, we propose that SCR activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity. This study characterizes a combinatorial interaction between two HOX proteins; a mechanism that may have a major role in patterning the anterior-posterior axis of other animals.

Animals↗

Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin).

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the mu-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.

Adaptor Proteins, Vesicular Transport↗

Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus.

Triclosan, a widely used antibacterial agent, possesses potent activity against Staphylococcus aureus. This study reports on an investigation of the antibacterial target of triclosan in this pathogen. A strain of S. aureus overexpressing the enoyl-[acyl-carrier-protein] reductase (FabI), demonstrated by Western immunoblotting, gave rise to an increase in the MIC of triclosan, while susceptibilities to a range of unrelated antibacterials were unaffected. There are approximately 12 000 molecules of FabI per cell in mid-log phase growth. This number increased by approximately three- to four-fold in the S. aureus FabI overexpressor. Triclosan selectively inhibited the incorporation of [(14)C]acetate into TCA-precipitable product, an indicator of fatty acid biosynthesis. Furthermore, it inhibited de novo fatty acid biosynthesis in this organism. In vitro, triclosan inhibited recombinant, purified S. aureus FabI with an IC(50) of approximately 1 microM. The combination of these biochemical and genetic data provide further evidence that the mode of action of triclosan in S. aureus is via inhibition of FabI.

Anti-Infective Agents, Local↗

Genetic characterization of the multidrug-resistant phenotype of VM-26-resistant human leukemic cells.

Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone. However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987). More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988). These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR). In the present work, we studied the somatic cell genetics of at-MDR. We produced hybrid cell lines by polyethylene glycol-mediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity. Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay. The hybrid lines all contained approximately 2x DNA compared to unfused controls, indicating that the fusions were successful. The IC50 for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10(-8) M, and another was 76 x 10(-8) M. These data indicate that drug sensitivity was reconstituted by the hybridization procedure. By comparison, the VM-26 IC50 values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10(-8) M, respectively. To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation"). Using 32P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 microM VM-26 maximally stimulated DNA cleavage by approximately 11-fold compared to no-drug controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Culture Media↗

Genetic characterization of single cell clones isolated from a radiation induced lymphoma in mice carrying the T (1:17) 190 chromosome translocation.

Single cell clones were isolated in vivo from early passages of a highly variable lymphoma induced by gamma-irradiation in a TF-T190/+ mouse carrying the T/1:17/T190Ca chromosome translocation. The chromosome pattern, expression of the H-2 antigenic specificities determined by the T190 (1:17) translocation chromosomes and biological properties of the clones have been compared and discussed.

Animals↗

Genetic characterization of rabies field isolates from Thailand.

We sequenced 512 nucleotides in two variable regions of the N gene of 23 rabies isolates from the northeastern part of Thailand by direct sequencing of PCR-amplified products. The sequencing data revealed two new lineages in these rabies isolates. Based on the results of this study together with the findings of our earlier study, the rabies isolates in Thailand were divided into two genogroups, designated as T1 and T2, which were predominantly localized in the northern and northeastern areas, respectively. Each of these two genogroups consisted of four lineages. There was a correlation between these eight lineages and the geographical origins of the isolates. Two lineages belonging to the T2 genogroup from the northeastern area of Thailand were newly identified in this study. The isolates in both genogroups were also prevalent in the central area of Thailand. Each lineage in the T1 and T2 genogroups was found independently in dogs in the upper and lower southern areas of Thailand, respectively. These genetic data and the historical background suggest that rabies viruses belonging to the T2 genogroups were prevalent many years ago in the central and northeastern areas of Thailand and were later transferred to the lower southern parts of Thailand.

Amino Acid Sequence↗

Genetic characterization of the body attributed to the evangelist Luke.

Historical sources indicate that the evangelist Luke was born in Syria, died in Greece, and then his body was transferred to Constantinople, and from there to Padua, Italy. To understand whether there is any biological evidence supporting a Syrian origin of the Padua body traditionally attributed to Luke, or a replacement in Greece or Turkey, the mtDNA was extracted from two teeth and its control region was cloned and typed. The sequence determined in multiple clones is an uncommon variant of a set of alleles that are common in the Mediterranean region. We also collected and typed modern samples from Syria and Greece. By comparison with these population samples, and with samples from Anatolia that were already available in the literature, we could reject the hypothesis that the body belonged to a Greek, rather than a Syrian, individual. However, the probability of an origin in the area of modern Turkey was only insignificantly lower than the probability of a Syrian origin. The genetic evidence is therefore compatible with the possibility that the body comes from Syria, but also with its replacement in Constantinople.

Base Sequence↗

Detection and genetic characterization of norovirus strains circulating among infants and children with acute gastroenteritis in Japan during 2004-2005.

A total of 752 fecal specimens collected during the period of July 2004 to June 2005 from infants and children with acute gastroenteritis from four different regions (Maizuru, Tokyo, Sapporo, and Osaka) of Japan were tested for the presence of norovirus by RT-PCR. It was found that 139 (18.5%) fecal specimens were positive for norovirus. Norovirus infection was detected almost all year round with the highest prevalence in January. Norovirus GII was the most predominant genogroup (98.6%; 137 of 139). The genotypes detected in this study were GI/1, GII/1, GII/3, GII/4, and GII/6. Of these, NoV GII/4 (known as the Lordsdale virus cluster) was re-emerging and became the leading genotype (77.7%). Meanwhile, the incidence of NoV GII/3 (known as the Arg320 virus cluster) has dropped rapidly, accounting for only 15.8%. Another interesting feature of the study was the identification of Picton03/AU-like recombinant NoV for the first time in Japan. Based on the genetic analysis, it was interesting to note that NoV GII/4 in 2004-2005 made a distinct cluster in comparison to other NoV GII/4 circulating in 2002-2003 and 2003-2004. Of note, "new recombinant variant designated GIIb" within NoV GII/3, which was first detected in Saga City, Japan in 2003-2004 in only one case, had increased, spreading widely in Japan and representing 45.5% (10 of 22). Further epidemiological studies should be conducted to determine whether this new recombinant variant strain will be dominant in Japan in the coming year.

Acute Disease↗

Genetic characterization of a large, historically significant Utah kindred with facioscapulohumeral dystrophy.

In 1950, Tyler and Stephens reported a remarkable kindred affected with facioscapulohumeral dystrophy (FSHD), consisting of 1249 descendants of a man who emigrated to Utah in 1840. Members of this kindred are still seen in our clinic and, to our knowledge, no member had been tested for deletions at the FSHD1A locus on chromosome 4q35, the common chromosomal rearrangement associated with FSHD. We have identified 971 additional members of this kindred who either were not included in or unborn at the time of the report by Tyler and Stephens, and have identified 120 living members as affected by history or by examination. Members of this kindred contribute to a disease prevalence of nearly 1:15 000 in the Utah/southern Idaho region. We have demonstrated that affected members carry a disease-associated 20 kb deletion allele at the FSHD1A locus. This allele is the same size in multiple, distantly-related branches of the kindred, confirming the meiotic stability of the FSHD1A deletion. This large, genetically homogeneous population of patients represents a unique resource with which to study current questions about FSHD, including the possibilities of anticipation and parental transmission effects.

Chromosomes, Human, Pair 4↗

Serological and genetic characterization of bovine rotaviruses in Thailand by ELISA and RNA-RNA hybridization: detection of numerous non-serotype 6 strains.

A total of 62 fecal specimens positive for rotavirus were collected from diarrheic cows in Thailand in 1988 and 1989. The antigenic properties of rotaviruses in stool were examined by enzyme-liked immunosorbent assays using specific monoclonal antibodies directed at VP4, VP6 or VP7: all the bovine rotavirus strains were determined as subgroup I; none of the strains were reactive with serotype 6-specific monoclonal antibody; and different reactivities of the bovine strains with two anti-VP4 monoclonal antibodies were observed. Polyacrylamide gel electrophoresis of viral RNA exhibited three different RNA electropherotypes. In RNA-RNA hybridization experiments using cell culture-adapted three strains as well as a reference bovine strain (NCDV), RNA from the Thai bovine strains showed very low homology to that from NCDV; only three or four RNA segments were hybridized between the RNAs from Thai samples and NCDV. These results suggested that bovine rotaviruses isolated in Thailand are serologically and genetically distinct from a reference serotype 6 bovine strain, NCDV.

Animals↗

Isolation of simian immunodeficiency viruses from two sooty mangabeys in Côte d'Ivoire: virological and genetic characterization and relationship to other HIV type 2 and SIVsm/mac strains.

OBJECTIVE: To search for the presence of SIV in sooty mangabeys and other monkey species in Côte d'Ivoire, West Africa, and to compare viral isolates with HIV-2 strains from the same region. METHODS: Forty-three captive housed monkeys (28 African green monkeys, 6 sooty mangabeys, 6 baboons, and 6 patas monkeys) were tested for the presence of HIV and SIV antibodies. Virus was isolated from the peripheral blood lymphocytes of seropositive animals and from HIV-2 antibody-positive patients originating from Côte d'Ivoire, Ghana, Senegal, and Belgium. Viruses were characterized by Western blot and radioimmunoprecipitation assay. Proviral DNA was amplified by PCR, cloned, and sequenced to construct a phylogenetic tree. RESULTS: One African green monkey and three sooty mangabeys had antibodies that cross-reacted with HIV-2. From two mangabeys lentiviruses were isolated and designated as SIVsmCI2 and SIVsmCI8. Serological, virological, and sequence data showed that these isolates are members of the HIV-2/SIVsm/SIVmac group of primate lentiviruses. Furthermore, in the phylogenetic tree, these two new viruses form a distinct subgroup that is equidistant to the HIV-2 strains and the previously described SIVsm/SIVmac viruses. CONCLUSION: This study provides additional evidence that sooty mangabey monkeys can be infected with a lentivirus in their natural habitat. Within the SIVsm and SIVmac viruses extensive genetic variation is observed.

Acquired Immunodeficiency Syndrome↗

Physiological and genetic characterization of hydrotropic mutants of Arabidopsis thaliana.

Roots display positive hydrotropism in response to moisture gradient. Hydrotropism regulates the directional growth by interaction with other growth movements. Using the seedlings of pea, cucumber, maize and wheat, we have revealed that the root cap perceives the moisture gradient and that auxin and calcium are involved in hydrotropism. However, molecular mechanisms for stimulus perception or signal transduction in hydrotropism are still remained unrevealed. To dissect the molecular mechanism underlying hydrotropism in seedling roots, we established a method for screening Arabidopsis mutants defective in root hydrotropism. Among about 20,000 M2 seedlings of Arabidopsis plants treated with EMS, we successfully obtained 12 mutants of which root hydrotropism was reduced to various extents. We named them root hydrotropism (rhy) and examined their gravitropism, phototropism, waving response and elongation growth as well as hydrotropism in roots. Roots of rhy1 mutant showed ahydrotropic response although the other responses and elongation growth of rhy1 mutant were normal. Roots of rhy2 and rhy3 mutants showed a reduced hydrotropism and abnormal responses in gravitropism, phototropism or waving pattern. Genetic analysis of the progeny produced by the backcross of rhy1 mutant to wild type suggested that rhy1 was a recessive mutation. We also examined the map position of the rhy1 locus.

Arabidopsis↗

Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building.

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5'-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phe139Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Arg169Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Amino Acid Sequence↗

Genetic characterization of biodiversity in highly inbred chicken lines by microsatellite markers.

Forty-two microsatellite loci were analysed in 23 highly inbred chicken lines derived from Leghorn, Jungle Fowl, Fayoumi and Spanish breeds. Line-specific alleles among breeds and lines were detected. The band-sharing (BS) values were calculated and the proportion of shared alleles distances (Dps) were estimated. The BS values and Dps between sets of MHC-congenic lines ranged from 0.74 to 0.96, and 0.05-0.35, respectively. The BS values between each pair of noncongenic Leghorn lines were 0.32-0.97, and between Leghorn and exotic (Jungle Fowl, Fayoumi and Spanish) breeds were 0.03-0.55. The Dps between Fayoumi lines and other lines were much larger (0.66-1.34) than within Leghorns, and the Jungle Fowl breed had the largest distances with other lines (1.12-5.38). The phylogenetic consensus tree that was constructed grouped these 23 inbred chicken lines into four different clusters. These results are in accordance with the origin and breeding history of these inbred lines, which indicates that the use of microsatellites for the study of genetic biodiversity is accurate and reliable. In addition, the significance and value of inbred chicken lines in molecular genetic research is discussed.

Alleles↗

Cytogenetic and molecular genetic characterization of trisomy 20 mosaicism in fetal blood and tissues.

We report a case of mosaic trisomy 20, the most common autosomal mosaicism identified in amniocytes, ascertained in a woman referred for amniocentesis because of abnormal ultrasound at 18.1 weeks' gestation which revealed short femurs and nuchal thickening. Metaphase analysis of 98 clones revealed 47,XY, +20 in 96 cells (98 per cent). Trisomy 20 was demonstrated in 6 cells (12 per cent) in a total of 50 cells from two fetal blood cultures obtained after pregnancy termination. Fluorescence in situ hybridization (FISH) analysis of interphase nuclei utilizing a chromosome 20 alpha-satellite centromeric DNA probe revealed three signals in 57/546 nuclei (10 per cent) in fetal blood. Metaphase analysis of 167 cells from seven different fetal tissue sources revealed trisomy 20 in 32 cells (19.2 per cent). The percentage of trisomy 20 cells varied with tissue type, with the highest percentage (13/25 cells, 52 per cent) identified in the small intestine and lymph nodes and the lowest percentage (1/34 cells, 2.9 per cent) identified in a specimen of chorionic villi. Molecular genetic analyses utilizing polymerase chain reaction (PCR)-formated dinucleotide repeat polymorphisms demonstrated that the non-disjunctional event most likely occurred post-zygotically and that the origin of the extra chromosome 20 was maternal. This study is the first to demonstrate trisomy 20 cells in fetal blood, suggesting that mosaic trisomy 20 can be embryonic in origin. In cases of prenatally detected mosaic trisomy 20, examination of fetal blood should be considered, as well as study of placental membranes, skin, and urine sediment to confirm the karyotype and determine its significance.

Adult↗

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants.

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.

Bacillus megaterium↗

Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclase.

Sixty-two spontaneous mutations have been characterized which reduce the level of expression of catabolite-sensitive operons. These mutations appear to affect either the crp (catabolite gene activator protein) or cya (adenyl cyclase) loci. No new loci have been discovered. Deletions of the cya gene do not remove an essential function. phi80 transducing phage for the cya gene have been used to do recombination and complementation studies on cya mutants.

Adenylyl Cyclases↗