Phlorizin and Trilobatin,Sweet Dihydrochalcone-Glucosides from Leaves of Lithocarpus litseifolius (Hance) Rehd. (Fagaceae)

To determine the primary structure of the α-amylase produced by Bacillus subtilis var. amylosacchariticus, we have reported the isolation of thirty-four tryptic peptides and eight CNBr fragments from the enzyme. Since the alignment of the eight CNBr fragments was made by matching with six methionine-containing tryptic peptides, the order of tryptic peptides within each CNBr fragment was determined. In the case of four small CNBr fragments, sequence analyses using an automated sequence analyzer established the peptide orders within these fragments. For larger fragments, further fragmentation was done using chymotrypsin or staphylococcal protease V8 and the resultant peptides were isolated and sequenced. Consequently, the peptide orders within three out of four large CNBr fragments were established.     

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.     

Complete amino acid sequence of copper-zinc superoxide dismutase from Drosophila melanogaster

The complete amino acid sequence of the Drosophila melanogaster Cu,Zn superoxide dismutase subunit has been determined by automated Edman degradation. Sequence analyses were performed on the intact S-carboxymethylated protein, two fragments derived from CNBr cleavage, and three peptides recovered from mouse submaxillary protease digestion of the reduced and S-carboxymethylated enzyme. The peptides were aligned by characterizing peptides yielded by trypsin and Staphylococcus aureus V8 protease. All the peptides studied were purified exclusively by reverse-phase columns of HPLC and were analyzed with an improved liquid-phase sequencer. A molecular weight of 15,750 (subunit) was calculated from the 151 residues sequenced. The amino acid sequence of the Drosophila superoxide dismutase subunit is compared with that of four other eucaryotes: man, horse, cow, and yeast. Comparison of the five primary structures reveals very different rates of evolution at different times. Copper-zinc superoxide dismutase appears to be a very erratic evolutionary clock. Val-Val-Lys-Ala- Val-Cys-Val-Ile-Asn-Gly-Asp-Ala-Lys-Gly-Thr-Val-Phe-Phe-Glu-Gln- Glu-Ser-Ser-Gly-Thr-Pro-Val-Lys-Val-Ser-Gly-Glu-Val-Cys-Gly-Leu- Ala-Lys-Gly-Leu-His-Gly-Phe-His-Val-His-Glu-Phe-Gly-Asp-Asn-Thr- Asn-Gly-Cys-Met-Ser-Ser-Gly-Pro-His-Phe-Asn-Pro-Tyr-Gly-Lys-Glu- His-Gly-Ala-Pro-Val-Asp-Glu-Asn-Arg-His-Leu-Gly-Asp-Leu-Gly-Asn- Ile-Glu-Ala-Thr-Gly-Asp-Cys-Pro-Thr-Lys-Val-Asn-Ile-Thr-Asp-Ser- Lys-Ile-Thr-Leu-Phe-Gly-Ala-Asp-Ser-Ile-Ile-Gly-Arg-Thr-Val-Val-Val- His-Ala-Asp-Ala-Asp-Asp-Leu-Gly-Gln-Gly-Gly-His-Glu-Leu-Ser-Lys- Ser-Thr-Gly-Asn-Ala-Gly-Ala-Arg-Ile-Gly-Cys-Gly-Val-Ile-Gly-Ile- Ala-Lys.     

NADPH-cytochrome P-450 reductase (pig liver). Studies on the sequence of the cyanogen bromide peptides from the catalytic domain and on the reactivity of the thiol groups

The reactivity of the cysteine residues in the non-denatured catalytic domain of the NADPH-cytochrome P-450 reductase (pig liver) was studied using the -SH reagent monobromobimane. Prerequisite was the characterization of the cysteine residues by their surrounding amino-acid sequences. In pursuit of these aims the CNBr fragments obtained from the catalytic domain were sequenced. The cysteine residues are distributed on six CNBr fragments of the catalytic domain [Vogel and Lumper (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 1074]. Only the 11-kDa CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr- and Ala-Ser-Ser-Ser-, respectively, contain two cysteine residues each. The cysteine residues of the catalytic domain accessible to monobromobimane were localized on three CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser- and Ala-Arg-Asp-Val-, respectively. Inactivation of the trypsin-solubilized enzyme by -SH-directed reagents is caused by the modification of the accessible cysteine residue (which can be protected by NADPH) in the 11-kDa CNBr fragment (N-terminal sequence: Val-Gly-Pro-Thr-). The cosubstrate NADPH protected a second cysteine residue localized in the 11-kDa CNBr peptide with the N-terminal sequence Ala-Ser-Ser-Ser-, which is however modified at a distinctly slower rate than the critical cysteine residue characterized by the sequence -Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg-Arg. Five non-reacting thiol groups were localized on CNBr fragments with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser-, Ser-Leu-Asn-Asn-, Gly-Lys-Tyr-Val-Asp- and Ala-Ala-Asp-Pro-.     

Synthesis of Penicillins and Cephalosporins by Penicillin Acylase Entrapped in Fibres

Tryptic peptides from two cyanogen bromide (CNBr) fragments CB II and CB III of the Ala chain of ricin D were sequenced by manual Edman degradation. Chymotryptic or peptic peptides from the two fragments were isolated by Dowex 1 x 2 column chromatography to obtain overlaps for the tryptic peptides, and the complete amino acid sequences of fragments CB II and III were established. The amino acid residues in fragments CB II and CB III accounted for 75 and 45 residues, respectively, of 260 residues in the Ala chain.

These sequences together with the sequence of fragment CBI described in the preceding paper established the complete sequence of the 260 amino acid residues in the Ala chain. Some structural characteristics of the protein are also discussed.     

The amino acid sequence of plastocyanin from Lactuca sativa (lettuce)

The amino acid sequence of plastocyanin from lettuce (Lactuca sativa L.) was determined by using a Beckman 890C automatic sequencer and by dansyl-phenylisothiocyanate analysis of peptides obtained by enzymic digestion of purified CNBr fragments. The protein consists of a single polypeptide chain of 99 residues, and shows close homology with other higher plant plastocyanins. The data are discussed in relation to the possible residues involved in the binding of copper in plastocyanin.     

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). II. The amino acid sequence and alignment of cyanogen bromide fragments IV, III, and I.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is the major protein constituent of the human plasma high density lipoproteins. Cleavage of this protein with cyanogen bromide yields four fragments, designated in the order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the first paper in this series, the amino acid sequence of the NH2-terminal fragment, CNBr II, is reported. In the present study, CNBr IV, III, and I, containing, respectively, 25, 36, and 94 amino acids were sequenced by conventional means. To establish the alignment of the cyanogen bromide fragments, apoLP-Gln-I was digested with trypsin and two of the three methionine-containing tryptic peptides were isolated. The amino acid sequence of apoLP-Gln-I is as follows: (see article). With the complete amino acid sequence available, a CPK space-filling model of apoLP-Gln-I was constructed. The protein was placed into an alpha helical conformation wherever the primary structure permitted. Thirteen helical regions were identified. These regions account for 70% of the amino acid residues of the protein. Each helix is characterized as having two faces (amphipathic). One is a polar face that occupies approximately 180 degrees of the surface of the helix and the other is a hydrophobic face that occupies the other 180 degrees of the helical surface. Similar amphipathic helices have been identified previously in the other lipoprotein-proteins that have known sequences. It is suggested that the amphipathic helical regions of apoLP-Gln-I are important in the binding of phospholipids in high density lipoproteins.     

Covalent structural analysis of yeast inorganic pyrophosphatase: Location of groups implicated in the catalytic function; placement of the NH2-terminal 100 residues in the subunit

Covalent structural analysis of two of the three cyanogen bromide fragments from yeast inorganic pyrophosphatase (EC 3.6.1.1, pyrophosphate phosphohydrolase) was undertaken by a strategy involving both automated Edman degradation and conventional sequence analysis. Automated degradation of intact, reduced and carboxymethylated pyrophosphatase provided the sequence of the first 34 residues in the NH₂-terminal 45-residue peptide, CNBr VI, in addition to a partial sequence through 50 cycles which confirmed the overlap into the internal fragment, CNBr III. The sequence of CNBr VI was completed through analysis of peptides derived from hydrolysis of the fragment with trypsin and chymotrypsin. Structural analysis of CNBr III has provided the sequence of the first 55 amino acids in this 103-residue fragment. The sequence was established by conventional and automated procedures applied to the analysis of tryptic peptides generated from the citraconylated fragment. These findings constitute the sequence of the first 100 residues in the pyrophosphatase subunit and, together with structural information obtained earlier, define over half of the covalent structure of the molecule. Moreover, the sequence derived thus far permits the placement of a number of amino acids that are of importance relative to studies of the enzyme mechanism, and with regard to analysis of its three-dimensional structure.     

The covalent structure of calf skin type III collagen. I. The amino acid sequence of the amino terminal region of the alpha 1(III) chain (position 1--222).

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4.

The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.     

The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.     

Primary structure of a human IgA1 immunoglobulin. II. Isolation, composition, and amino acid sequence of the tryptic peptides of the whole alpha1 chain and its cyanogen bromide fragments.

As part of the strategy for determining the covalent structure of a human IgA1 molecule (Bur), a tryptic digest was prepared of the reduced and carboxymethylated alpha1 heavy chain. In addition to the main experiment, tryptic peptides were prepared from the succinylated aminoethylated alpha1 chain and from fragments obtained by CNBr scission of the alpha1 chain. Complete recovery of the peptides was impeded by the large size of some of the tryptic peptides and of the principal CNBr fragment, and difficulty in separating other glycopeptides. Twenty-eight tryptic peptides of the reduced and carboxymethylated alpha1 chain were purified and sequenced, accounting for more than 300 residues. Additional information was obtained by sequence analysis of trypudies described in this series of papers contributed to the complete sequence analysis of the alpha1 chain.     

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.     

Amino acid sequence of alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii

The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented. The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I). The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37,652. The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today. Of these, only 18 residues (5%) are strictly conserved: 11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.     

The primary structure of the psychrophilic lactate dehydrogenase from Bacillus psychrosaccharolyticus

L-lactate dehydrogenase of the psychrophilic bacterium B. psychrosaccharolyticus was isolated by a three-step procedure and its total amino-acid sequence determined by automated Edman degradation. The protein consists of 318 amino-acid residues and its calculated molecular mass is 35,254 Da. Most of the primary structure could be established by sequencing large peptide fragments obtained by chemical cleavages, namely with BNPS-skatole and with CNBr. Further fragmentations of two tryptophan peptides with the endoproteinase Lys-C and with diluted HCl resulted in shorter overlapping peptides, the analysis of which completed the sequence. The C-terminal sequence Glu-Gln was established by carboxypeptidase A experiments and was then verified by the analysis of short C-terminal tryptic and chymotryptic peptides. The first lactate dehydrogenase sequenced so far of a psychrophilic bacillus shows sequence homologies between 60% and 75% to the enzymes from the mesophilic B. megaterium and B. subtilis and the thermophilic B. stearothermophilus, B. caldolyticus and B. caldotenax. Within the 50 N-terminal residues, three additional sequences could be included in our comparisons. In this part of the molecule, sequence homologies between 56% and 74% were calculated.     

Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments

The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.     

Amplification of ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) gene fragments from Thiobacillus ferrooxidans and a moderate thermophile using polymerase chain reaction

Southern blot analysis of DNA from an iron-oxidising moderate thermophile NMW-6 and from Thiobacillus ferrooxidans strain TF1–35 demonstrated sequences homologous to the RuBisCO LSU gene of Synechococcus. DNA fragments (457 bp) encoding part of the RuBisCO LSU gene (amino acids 73–200) were amplified from the genomic DNA of Thiobacillus ferrooxidans and the moderate thermophile NMW-6 using the polymerase chain reaction (PCR) technique (Saiki et al. (1985) Science 233, 1350–1354). A comparison with the LSU sequences from T. ferrooxidans, Alcaligenes eutrophus, Chromatium vinosum, Synechococcus and Spinacea oleracea, which all have RuBisCOs with a hexadecameric structure, showed that the RuBisCO LSU gene sequence from NMW-6 appeared to be most closely related to that of the hydrogen bacterium A. eutrophus which showed 71.9% homology at the amino acid level. Despite its physiological similarity, T. ferrooxidans showed only 64.1% homology to the amino acid sequence from NMW-6 and had the lowest DNA homology (60.9%) of the hexadecameric type RuBisCOs. In the region sequenced, T. ferrooxidans and the RuBisCOs of the phototrophs C. vinosum, Synechococcus and S. oleracea, had 17 residues that were completely conserved which were substituted in both NMW-6 and A. eutrophus, 11 of these being identical substitutions. Comparison of the nucleotide and derived amino acid sequences of the RuBisCO LSU fragment from T. ferrooxidans with other RuBisCO sequences indicated a closer relationship to the hexadecameric type LSU genes of photosynthetic origin than to that of A. eutrophus. The T. ferrooxidans amino acid sequence showed 93.8%, 78.9% and 77.3% homology, respectively, to the C. vinosum, Synechococcus and S. oleracea (spinach) sequences but only 56.2% to A. eutrophus. The DNA sequence from Rhodospirillum rubrum, which has the atypical large subunit dimer RuBisCO structure with no small subunit, showed 39.2% and 42.7% homology, respectively, with the sequences of NMW-6 and T. ferrooxidans, and 25.0% and 29.7% amino acid homology, indicating that the DNA homology was substantially random in nature. PCR fragments (126 bp) that overlaped the last 15 codons of the fragments above were also amplified and sequenced. They showed incomplete homology with the larger fragments, supporting evidence obtained from Southern hybridizations that T. ferrooxidans and the moderate thermophile NMW-6 have multiple copies of RuBisCO LSU genes.