Influence of Cytochrome P450, ABC and SLC Gene Polymorphisms on Imatinib Therapy Outcome of Patients with Gastrointestinal Stromal Tumours (GIST).

The efficacy of imatinib-based therapy depends on the proteins involved in its metabolism and transportation. Therefore, the aim of our study was to investigate the possible correlation of selected P450, ABC and SLC polymorphic variants and the outcome of imatinib therapy. A total of 101 patients with advanced, KIT/PDGFRA(+) GIST treated with imatinib were enrolled to the study. DNA was extracted from peripheral blood samples and genotypes were determined by PCR-RFLP and direct sequencing. Deviation from the Hardy-Weinberg equilibrium was only observed for rs2740574. None of the studied SNPs was associated with GIST time to progression. No significant correlation between any specific variant and time to progression was found in the group with KIT exon 11 mutation. However, individuals of at least three potentially unfavourable genotypes presented significantly shorter time to progression in comparison to patients with two or less unfavourable genotypes.

The analysis of the outcomes and factors related to iliac-obturator involvement in cutaneous melanoma patients after lymph node dissection due to positive sentinel lymph node biopsy or clinically detected inguinal metastases.

BACKGROUND: We assessed clinical-pathological features and outcomes of cutaneous melanoma patients after ilio-inguinal lymph node dissection (LND) in relation to the presence of metastases in iliac-obturator nodes. METHODS: We analyzed 390 consecutive patients who underwent ilio-inguinal therapeutic LND [TLND] (237) due to clinical/cytologically detected metastases or after completion LND [CLND] (153) due to positive SLN biopsy (in one cancer centre 1994-2009). Median follow-up time was 60 months. RESULTS: The 5-year overall survival (OS) rate was 49% and median OS - 52 months in the entire group of patients. According to univariate analysis following factors had significant negative influence on OS: presence of metastases to iliac-obturator nodes (5-year OS for positive versus negative: 54.5% and 32%, respectively), macrometastases, higher Breslow thickness, ulceration, higher Clark level, male gender, number of metastatic lymph nodes, extracapsular extension, and, additionally in the CLND group - micrometastases size ≥ 0.1 mm according to the Rotterdam criteria and non-subcapsular location of micrometastases. Iliac-obturator involvement was also negative factor for OS in multivariate analysis. The presence of iliac-obturator nodal metastases correlated with the following factors: type of LND-CLND versus TLND (15% versus 27.5%) of iliac-obturator involvement, respectively), higher Breslow thickness, extracapsular extension of nodal metastases, male gender. We have not identified any metastases in iliac-obturator nodes in group of patients with micrometastases size ≤1.0 mm and primary tumour Breslow thickness <4.0 mm or no ulcerated primary tumours. CONCLUSIONS: Metastases to iliac-obturator nodes have additional negative prognostic value for melanoma patients with inguinal basin involvement. We are able to identify the subgroup of patients after positive SLN biopsy without metastases to iliac-obturator nodes, probably requiring only inguinal LND.

Clinical, pathological and surgical characteristics of duodenal gastrointestinal stromal tumor and their influence on survival: a multi-center study.

BACKGROUND: The duodenum is a rare site of primary gastrointestinal stromal tumor (GIST). Overall (OS) and disease-free survival (DFS) after limited resection (LR) versus pancreaticoduodenectomy (PD) were studied. METHODS: All patients who underwent surgery for primary, localized duodenal GIST between 2000 and 2011 were identified from four prospective institutional databases. OS and DFS were calculated by Kaplan-Meier method. Univariate analysis was performed. RESULTS: Eighty-four patients (median follow-up 42 months) underwent LR (n = 56, 67%) or PD (n = 28, 33%). Patients in the PD group had a larger median tumor size (7 cm vs. 5 cm, p = 0.024) and higher mitotic count (39% vs. 19% >5/50 high-power fields, p = 0.05). Complications were observed in five patients (9%) in the LR group and ten patients (36%) in the PD group. OS and DFS for the entire cohort were 89% and 64% at 5 years, respectively. No difference in outcome between LR and PD were observed. Eleven patients were treated with preoperative IM. A major RECIST response was obtained in nine (80%), whereas two had stable disease. Twenty-three patients received postoperative Imatinib (IM). A trend toward a better OS in IM-treated patients could be detected only in the high-risk group. CONCLUSIONS: Type of duodenal resection does not impact outcome. The choice should be determined by duodenal site of origin and tumor size. IM may be considered in cases at high risk of recurrence; in neoadjuvant setting, IM might facilitate resection and possibly increase the chance of preserving normal biliary and pancreatic anatomy.

Prognostic value of KIT/PDGFRA mutations in gastrointestinal stromal tumours (GIST): Polish Clinical GIST Registry experience.

BACKGROUND: Majority of gastrointestinal stromal tumours (GISTs) are characterised by KIT-immunopositivity and the presence of KIT/platelet-derived growth factor receptor alpha (PDGFRA) activating mutations. PATIENTS AND METHODS: Spectrum and frequency of KIT and PDGFRA mutations were investigated in 427 GISTs. Univariate and multivariate analysis of relapse-free survival (RFS) was conducted in relation to tumours' clinicopathologic features and genotype. RESULTS: Mutations were found in 351 (82.2%) cases, including 296 (69.3%) KIT and 55 (12.9%) PDGFRA isoforms. Univariate analysis revealed higher 5-year RFS rate in women (37.9%; P = 0.028) and in patients with gastric tumours (46.3%; P < 0.001). In addition a better 5-year RFS correlated with smaller tumour size ≤ 5 cm (62.7%; P < 0.001), tumours with mitotic index ≤ 5/50 high-power fields (60%; P < 0.001), and characterised by (very) low/moderate risk (70.2%; P = 0.006). Patients with GISTs bearing deletions encompassing KIT codons 557/558 had worse 5-year RFS rate (23.8%) than those with any other KIT exon 11 mutations (41.8%; P < 0.001) or deletions not involving codons 557/558 (33.3%; P = 0.007). Better 5-year RFS characterised patients with KIT exon 11 point mutations (50.7%) or duplications (40%). By multivariate analysis, tumours with PDGFRA mutations and KIT exon 11 point mutations/other than 557/558 deletions had lower risk of progression than with KIT exon 11 557/558 deletions (both Ps = 0.001). CONCLUSIONS: KIT/PDGFRA mutational status has prognostic significance for patients' outcome and may help in management of patients with GISTs.

Validation of the Joensuu risk criteria for primary resectable gastrointestinal stromal tumour - the impact of tumour rupture on patient outcomes.

BACKGROUND: Approval of imatinib for adjuvant treatment of gastrointestinal stromal tumours (GIST) raised discussion about accuracy of prognostic factors in GIST and the clinical significance of the available risk stratification criteria. METHODS: We studied the influence of a new modification of the NIH Consensus Criteria (the Joensuu risk criteria), NCCN-AFIP criteria, and several clinicopathological factors, including tumour rupture, on relapse-free survival (RFS) in a prospectively collected tumour registry series consisting of 640 consecutive patients with primary, resectable, CD117-immunopositive GIST. The median follow-up time after tumour resection was 39 months. None of the patients received adjuvant imatinib. RESULTS: The median RFS time after surgery was 50 months. In univariable analyses, high Joensuu risk group, tumour mitotic count >5/50 HPF, size >5 cm, non-gastric location, tumour rupture (7% of cases; P = 0.0014) and male gender had adverse influence on RFS. In a multivariable analysis mitotic count >5/50HPF, tumour size >5 cm and non-gastric location were independent adverse prognostic factors. Forty, 151, 86 and 348 patients were assigned according to the Joensuu criteria to very low, low, intermediate and high risk groups and had 5-year RFS of 94%, 94%, 86% and 29%, respectively. CONCLUSION: The Joensuu criteria, which include 4 prognostic factors (tumour size, site, mitotic count and rupture) and 3 categories for the mitotic count, were found to be a reliable tool for assessing prognosis of operable GIST. The Joensuu criteria identified particularly well high risk patients, who are likely the proper candidates for adjuvant therapy.

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: high-field EPR study.

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D+ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.

Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.

Comparison of a beta-glucosidase and a beta-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Purification, characterization, gene cloning, and sequence analysis.

Two distinct exo-acting, beta-specific glycosyl hydrolases were purified to homogeneity from crude cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus: a beta-glucosidase, corresponding to the one previously purified by Kengen et al. (Kengen, S. W. M., Luesink, E. J., Stams, A. J. M., and Zehnder, A. J. B. (1993) Eur. J. Biochem. 213, 305-312), and a beta-mannosidase. The beta-mannosidase and beta-glucosidase genes were isolated from a genomic library by expression screening. The nucleotide sequences predicted polypeptides with 510 and 472 amino acids corresponding to calculated molecular masses of 59.0 and 54.6 kDa for the beta-mannosidase and the beta-glucosidase, respectively. The beta-glucosidase gene was identical to that reported by Voorhorst et al. (Voorhorst, W. G. B., Eggen, R. I. L., Luesink, E. J., and deVos, W. M. (1995) J. Bacteriol. 177, 7105-7111; GenBank accession no. U37557U37557). The deduced amino acid sequences showed homology both with each other (46.5% identical) and with several other glycosyl hydrolases, including the beta-glycosidases from Sulfolobus solfataricus, Thermotoga maritima, and Caldocellum saccharolyticum. Based on these sequence similarities, the beta-mannosidase and the beta-glucosidase can both be classified as family 1 glycosyl hydrolases. In addition, the beta-mannosidase and beta-glucosidase from P. furiosus both contained the conserved active site residues found in all family 1 enzymes. The beta-mannosidase showed optimal activity at pH 7.4 and 105 degrees C. Although the enzyme had a half-life of greater than 60 h at 90 degrees C, it is much less thermostable than the beta-glucosidase, which had a reported half-life of 85 h at 100 degrees C. Km and Vmax values for the beta-mannosidase were determined to be 0.79 mM and 31.1 micromol para-nitrophenol released/min/mg with p-nitrophenyl-beta-D-mannopyranoside as substrate. The catalytic efficiency of the beta-mannosidase was significantly lower than that reported for the P. furiosus beta-glucosidase (5.3 versus 4, 500 s-1 mM-1 with p-nitrophenyl-beta-D-glucopyranoside as substrate). The kinetic differences between the two enzymes suggest that, unlike the beta-glucosidase, the primary role of the beta-mannosidase may not be disaccharide hydrolysis. Other possible roles for this enzyme are discussed.

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides.

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10. This may reflect some differences in the respective structures of both strains or, alternatively, may be due to deprotonation of TyrL 215 in Rb. capsulatus. At pH 7, measurements of the rate constant of QA to QB electron transfer reveal a threefold greater rate in the reaction centers from wild type Rb. capsulatus (65 +/- 1 0 ps)-1 compared to Rb. sphaeroides.We suggest that this may arise from a 0.7-A smaller distance between the quinones in the former strain. Our spectroscopic data on the wild type Rb. capsulatus reaction center suggest the existence of notable differences with the Rb. sphaeroides reaction center structure.

Effects of pigment-protein interactions on the conformation of the primary electron acceptor in Rhodobacter capsulatus reaction centers.

Resonance Raman spectra are reported for RCs from Rb. capsulatus in which the L104 glutamic acid is replaced by glutamine. The skeletal modes of the primary electron acceptor, BPhL, in these RCs undergo temperature-dependent frequency shifts that are identical to those observed for BPhL in RCs from wild-type. This observation suggests that the strength of the hydrogen bond between the L104 residue and the C9 keto group of BPhL is not a determinant of the temperature-dependent conformation of this pigment.

Resonance Raman studies of genetically modified reaction centers from Rhodobacter capsulatus.

Resonance Raman (RR) spectra are reported for the photosynthetic reaction center (RC) proteins from Rhodobacter capsulatus wild type and the genetically modified systems GluL104----Leu and HisM200----Leu. The spectra were obtained with a variety of excitation wavelengths, spanning the UV, violet, and yellow-green regions of the absorption spectrum, and at temperatures of 30 and 200 K. The RR data indicate that the structures of the bacteriochlorin pigments in RCs from Rb. capsulatus wild type are similar to those in RCs from Rhodobacter sphaeroides wild type. The data also show that the amino acid modifications near the primary electron acceptor (GluL104----Leu) and special pair (HisM200----Leu) perturb only those bacteriochlorin pigments near the site of the mutation and do not influence the structures of the other pigments in the RC. In the case of the GluL104----Leu mutant, elimination of the hydrogen bond to the C9 keto group of BPhL results in frequency shifts of RR bands of certain skeletal modes of the macrocycle. This allows the assignment of bands to the individual BPhL and BPhM pigments. In the case of the HisM200----Leu mutant, in which the special pair is comprised of a bacteriochlorophyll (BChl)-bacteriopheophytin (BPh) heterodimer rather than the BChl2 unit bound in the wild type, certain skeletal vibrations due to the additional BPh unit are identified. The frequencies of these modes are similar to those of the analogous vibrations BPhL and BPhM, which indicates that the structure of the BPh in the heterodimer is not unusual in any discernible way.(ABSTRACT TRUNCATED AT 250 WORDS)

EPR characterization of genetically modified reaction centers of Rhodobacter capsulatus.

Electron paramagnetic resonance (EPR) has been used to investigate the cation and triplet states of Rhodobacter capsulatus reaction centers (RCs) containing amino acid substitutions affecting the primary donor, monomeric bacteriochlorophylls (Bchls), and the photoactive bacteriopheophytin (Bphe). The broadened line width of the cation radical in HisM200----Leu and HisM200----Phe reaction centers, whose primary donor consists of a Bchl-Bphe heterodimer, indicates a highly asymmetric distribution of the unpaired electron over the heterodimer. A T0 polarized triplet state with reduced yield is observed in heterodimer-containing RCs. The zero field splitting parameters indicate that this triplet essentially resides on the Bchl half of the heterodimer. The cation and triplet states of reaction centers containing HisM200----Gln, HisL173----Gln, GluL104----Gln, or GluL104----Leu substitutions are similar to those observed in wild type. Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that are positioned near the central Mg2+ ions of the reaction center monomeric bacteriochlorophylls. Reaction centers containing serine substitutions at M180 and L153 or a threonine substitution at L153 have unaltered pigment compositions and are photochemically active. The cation and triplet states of HisL153----Leu reaction centers are similar to those observed in wild type. Triplet energy transfer to carotenoid is not observed at 100 K in HisM180----Arg chromatophores. These results have important implications for the structural requirements of tetrapyrrole binding and for our understanding of the mechanisms of primary electron transfer in the reaction center.

Stark effect in wild-type and heterodimer-containing reaction centers from Rhodobacter capsulatus.

The effect of an external electric field on the optical absorption spectra of wild-type Rhodobacter capsulatus and two Rb. capsulatus reaction centers that have been genetically modified through site-directed mutagenesis (HisM200----LeuM200 and HisM200----PheM200) was measured at 77 K. The two genetically modified reaction centers replace histidine M200, the axial ligand to the M-side bacteriochlorophyll of the special pair, with either leucine or phenylalanine. These substitutions result in the replacement of the M-side bacteriochlorophyll with bacteriopheophytin, forming a bacteriochlorophyll-bacteriopheophytin heterodimer. The magnitude of the change in dipole moment from the ground to excited state (delta mu app) and the angle delta between the Qy transition moment and the direction of delta mu app were measured for the special pair absorption band for all three reaction centers. The values for delta mu app and delta obtained for wild-type Rb. capsulatus (delta mu app = 6.7 +/- 1.0 D, delta = 38 +/- 3 degrees) were the same within experimental error as those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. The values for delta mu app and delta obtained for the red-most Stark band of both heterodimers were the same, but delta mu was substantially different from that of wild-type reaction centers (HisM200----LeuM200, delta mu app greater than or equal to 14.1 D and delta = 33 +/- 3 degrees; HisM200----PheM200, delta mu app greater than or equal to 15.7 D and delta = 31 +/- 4 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Directed mutations affecting spectroscopic and electron transfer properties of the primary donor in the photosynthetic reaction center.

Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that act as axial ligands to the central Mg(2+) ions of the "special pair" bacteriochlorophylls in the reaction center of Rhodobacter capsulatus. Histidine-173 of the L subunit has been replaced with glutamine, while histidine-200 of the M subunit has been replaced with glutamine, leucine, or phenylalanine. When leucine or phenylalanine is introduced at M200, one of the special pair bacteriochlorophylls is converted to bacteriopheophytin, which generates a heterodimer at the special pair binding site. The pigment composition of the reaction center is unaltered when either histidine is replaced with glutamine. All of these mutant reaction centers are photochemically active, although the electron transfer properties of heterodimer-containing reaction centers are altered. These mutations begin to define the structural parameters that determine whether bacteriochlorophyll or bacteriopheophytin will be incorporated into the tetrapyrrole binding sites of the photosynthetic reaction center. Our results demonstrate that the properties of the photosynthetic reaction center can be changed by directed mutagenesis, which makes this complex an excellent model for testing theories of electron transfer in biological systems.

Electron transfer in a genetically modified bacterial reaction center containing a heterodimer.

Time-resolved optical measurements encompassing the femtoseconds to seconds time scales have been used to investigate Rhodobacter capsulatus reaction centers (RCs) in which the histidine residue at position 200 on the M polypeptide has been changed to a leucine by site-directed mutagenesis. The HisM200----Leu RC, which has a heterodimer consisting of a bacteriochlorophyll and a bacteriopheophytin, is capable of the primary photochemistry observed in wild-type Rb. capsulatus RCs, but with an overall quantum yield reduced by about half. The lower yield resides in the initial electron transfer reaction and may be associated in part with substantial charge transfer character of the excited heterodimer. These and other comparisons between Rb. capsulatus wild-type and HisM200----Leu RCs have important implications for our understanding of the mechanism of electron transfer in the RC and the efficiency of the charge separation process.

Plasmid pU29, a vehicle for mutagenesis of the photosynthetic puf operon in Rhodopseudomonas capsulata.

Plasmid pU21, which carries the reaction center and light-harvesting genes (puf operon) of Rhodopseudomonas capsulata, has been redesigned by site-specific mutagenesis. Five restriction sites have been removed and three unique restriction sites have been introduced into this 11,589-bp pBR322 derivative. The modifications divide the puf structural genes into four regions separated by five unique and nonmutagenic restriction sites. These four fragments have been subcloned into the M13-mp series of vectors to facilitate oligonucleotide-mediated site-specific mutagenesis experiments on the photosynthetic apparatus structural genes. The inserts can then be returned from the M13 replicative form to the redesigned pU21 derivative. The modified plasmid, pU29, greatly facilitates in vitro mutagenesis experiments since previously described techniques and screening procedures are more efficient with M13 derivatives carrying smaller inserts. Additionally, tandem homologous sequences (the reaction center L and M subunits) within the puf operon are now separated on different phage vectors, eliminating problems encountered in the targeting of mutagenic oligonucleotides to only one of the two homologous sites.

Chromosomal deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from Rhodopseudomonas capsulata.

Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed wherein all or part of the reaction center (RC), light-harvesting I (LHI), and light-harvesting II (LHII) structural genes have been deleted. In one series of strains, the 2778-bp ApaI fragment bearing more than 90% of the rxcA operon (promoter and structural genes coding for LHI beta, LHI alpha, RC-L and RC-M) has been deleted from the chromosome. When the rxcA operon is deleted, resultant strains possess only LHII and are photosynthetically defective. The rxcA deletion in an LHII- background results in a strain lacking all LH antennae and RC subunits. As expected this strain has no near-infrared absorption characteristic of LH or RC bacteriochlorophyll. The rxcA deletion may be complemented by a pBR322 derivative carrying the entire rxcA operon (pU21). In a second series of deletion mutants, the 2500-bp BstEII-StuI fragment, including the beta and alpha structural genes coding for LHII has been deleted from the chromosome. In the wild-type background, functional RC and LHI are synthesized. LHII may be restored in the deletion strain by conjugal transfer of the plasmid pU2 which carries the LHII operon.

Nucleotide and deduced polypeptide sequences of the photosynthetic reaction-center, B870 antenna, and flanking polypeptides from R. capsulata.

The complete nucleotide sequence (8867 bp) and the deduced polypeptide sequence are given for 11 proteins from the photosynthetic gene cluster of R. capsulata (46 kb), including the photosynthetic reaction-center L, M, and H subunits and the B870 alpha and B870 beta polypeptides (light-harvesting I). These polypeptides bind bacteriochlorophyll, bacteriopheophytin, carotenoids, and quinones that are involved in the primary light reactions of photosynthesis. Hydropathy plots indicate that the L and M subunits are transmembrane proteins that may cross the membrane five times, while the H subunit has only one hydrophobic section near the amino terminus, which may be transmembrane. The L and M subunits are homologous over their entire length and have a high degree of homology with the QB protein from photosystem II of higher plants. An additional six genes were identified that may have some unknown role in bioenergetics since only mutations that affect the differentiation of the photosynthetic apparatus are known to map to this gene cluster.

Reaction center and light-harvesting I genes from Rhodopseudomonas capsulata.

Five structural genes coding for the reaction center (RC) L, M, and H subunits and the two light-harvesting (LH) I polypeptides, B870alpha and B870beta, have been mapped on two restriction fragments from the R-prime plasmid pRPS404. It has been recently shown that enhanced near-infrared fluorescence mutants of Rhodopseudomonas capsulata typically lack RC or LH I polypeptides and that these lesions are marker-rescued by two restriction fragments from the R-prime plasmid: the 7.5-kilobase-pair EcoRI F fragment and the 4.75-kilobase-pair BamHI C-EcoRI fragment. We have now determined the nucleotide sequence of two restriction fragments and have found that the BamHI C-EcoRI B fragment carries the structural genes for the RC L and M subunits and both LH I polypeptides. Forty kilobase pairs away from this locus, the BamHI F fragment (within the EcoRI F fragment) carries the RC H subunit. The structural genes on the BamHI C-EcoRI B fragment are probably transcribed as part of a polycistronic mRNA. All of the structural genes begin with a consensus Shine-Dalgarno sequence and separate AUG start codons, indicating that the structural polypeptides are not cleaved from larger precursor polypeptides.