Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium

The lux genes required for expression of luminescence have been cloned from a terrestrial bacterium, Xenorhabdus luminescens, and the nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits of luciferase determined. The lux gene organization was closely related to that of marine bacteria from the Vibrio genus with the luxD gene being located immediately upstream and the luxE downstream of the luciferase genes, luxAB. A high degree of homology (85% identity) was found between the amino acid sequences of the alpha subunits of X. luminescens luciferase and the luciferase from a marine bacterium, Vibrio harveyi, whereas the beta subunits of the two luciferases had only 60% identity in amino acid sequence. The similarity in the sequences of the alpha subunits of the two luciferases was also reflected in the substrate specificities and turnover rates with different fatty aldehydes supporting the proposal that the alpha subunit almost exclusively controls these properties. The luciferase from X. luminescens was shown to have a remarkably high thermal stability being stable at 45 degrees C (t 1/2 greater than 3 h) whereas V. harveyi luciferase was rapidly inactivated at this temperature (t 1/2 = 5 min). These results indicate that the X. luminescens lux system may be the bacterial bioluminescent system of choice for application in coupled luminescent assays and expression of lux genes in eukaryotic systems at higher temperatures.     

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.     

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.     

The effect of Clp proteins on DnaK-dependent refolding of bacterial luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK-DnaJ-GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution.     

Two distinct subunits of hemerythrin from the brachiopod Lingula reevii: an apparent requirement for cooperativity in O2 binding

Reported are results on the subunit composition of octameric hemerythrin (Hr) from the brachiopod Lingula reevii. Unlike most other Hrs, L. reevii Hr shows cooperativity in O2 binding. Purified L. reevii Hr was found to consist of two different subunits in approximately equimolar proportions. These two subunits differ in molecular weight by approximately 1000. Amino acid sequence data for the first 24 residues of the two subunits, labeled alpha and beta, show 70% identity with each other. Comparisons to amino acid sequences of other Hrs show approximately 50% identity in the first 24 residues and that both the alpha and beta subunits of L. reevii Hr have one residue deleted at their amino termini. Very recently, one other Hr, that from the brachiopod Lingula unguis, was also shown to contain equimolar proportions of two different subunits [Satake, K., Yugi, M., Kamo, M., Kihara, H., & Tsugita, A. (1990) Protein Seq. Data Anal. 3, 1-5], and this Hr also shows cooperativity in O2 binding. An alpha 4 beta 4 octamer is, therefore, proposed to be a common feature of those Hrs that show such cooperativity. Likely arrangements of alpha and beta subunits within an alpha 4 beta 4 octamer having the same configuration of subunits as that in other octameric Hrs are proposed. The most probable arrangements can be readily derived from physically reasonable restrictions on the types of intersubunit interactions and on transmission of allosteric effects.     

Control of luminescence decay and flavin binding by the LuxA carboxyl-terminal regions in chimeric bacterial luciferases.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generation of an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreum LuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted into a chimeric luciferase, LuxA(1)B, with a significantly more rapid decay rate. Two other chimeras with P. phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA(2)B and LuxA(3)B, retained the characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with both reduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependence of the luminescence decay on concentration and type of fatty aldehyde indicated that the decay rate of "fast" luciferases arose due to a high dissociation constant (K(a)) for aldehyde (A) coupled with the rapid decay of the resultant aldehyde-free complex via a dark pathway. The decay rate of luminescence (k(T)) was related to the decanal concentration by the equation: k(T) = (k(L)A + k(D)K(a))/(K(a) + A), showing that the rate constant for luminescence decay is equal to the decay rate via the dark- (k(D)) and light-emitting (k(L)) pathways at low and high aldehyde concentrations, respectively. These results strongly implicate the central region in LuxA(1)B as critical in differentiating between "slow" and "fast" luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.     

Molecular cloning and sequence analysis of aggretin, a collagen-like platelet aggregation inducer.

A cDNA library derived from the Malayan-pit-viper (Calloselasma rhodostoma) venom gland was constructed in the phagemid vector. Using the information of the N-terminal amino acid sequences of two subunits of aggretin, synthetic mixed-base oligonucleotides were employed as a screening probe for colony hybridization. Separate cDNA clones encoding for the alpha and beta chains of aggretin were isolated and sequenced. The results revealed that mature alpha and beta chains contain 136 and 123 amino acid residues, respectively. Aggretin subunits show high degrees of identity with respective subunits (50-60% for alpha, 49-58% for beta) of C-type lectin-like snake venoms. The identity to rattlesnake lectin is relatively lower (i.e., 39 and 30%). All cysteine residues in each chain of aggretin are well conserved and located at the positions corresponding to those of C-type lectins. Thus, three intracatenary disulfide bridges and an interchain disulfide bond between Cys83(alpha) and Cys75(beta) may be allocated. This is the first report regarding the entire sequence of venom GPIa/IIa agonist. According to the alignment of amino acid sequences, hypervariable regions among these C-type lectin-like proteins were revealed. These hypervariable regions are proposed to be the counterparts directly interacting with different receptors or different domains of a receptor on the surface of platelet.     

Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase alpha and beta subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate that neither the specific activity nor the size of the alpha (362 amino acid residues, Mr 41,389) and beta (324 amino acid residues, Mr 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the alpha and beta subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.     

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens.

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.     

Dynamic fluorescence study of the interaction of lumazine protein with bacterial luciferases

The equilibrium association of lumazine protein from Photobacterium phosphoreum with luciferases from either P. phosphoreum or an aldehyde-requiring dark mutant of Vibrio harveyi is measured from changes of the rotational correlation time which is derived from the decay of the lumazine ligand's fluorescence anisotropy. The rotational correlation time of lumazine protein is 23 ns (2 degrees C, 0.25 M Pi) and is increased on addition of luciferase due to the formation of a higher molecular weight complex. The V. harveyi luciferase exhibits full competence for the association and a 1:1 stoichiometry with a Kd in the range 40-90 microM. At lower ionic strength (0.05 M Pi), the Kd increases but is reduced again by the addition of dodecanol or dimyristoyllecithin. In contrast, tetradecanal, a substrate for the bioluminescence reaction, exerts no influence on the association. The equilibration rate is found to be too slow and for both luciferases the Kd values are too high for the interaction of the native proteins to account quantitatively for the spectral shifting of the bioluminescence by lumazine protein.     

Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo

Bacterial luciferase is a heterodimeric enzyme comprising two nonidentical but homologous subunits, alpha and beta, encoded by adjacent genes, luxA and luxB. The two genes from Vibrio harveyi were separated and expressed from separate plasmids in Escherichia coli. If both plasmids were present within the same E. coli cell, the level of accumulation of active dimeric luciferase was not dramatically less than within cells containing the intact luxAB sequences. Cells carrying the individual plasmids accumulated large amounts of individual subunits, as evidenced by two-dimensional polyacrylamide gel electrophoresis. Mixing of a lysate of cells carrying the luxA gene with a lysate of cells carrying the luxB gene resulted in formation of very low levels of active heterodimeric luciferase. However, denaturation of the mixed lysates with urea followed by renaturation resulted in formation of large amounts of active luciferase. These observations demonstrate that the two subunits, alpha and beta, if allowed to fold independently in vivo, fold into structures that do not interact to form active heterodimeric luciferase. The encounter complex formed between the two subunits must be an intermediate structure on the pathway to formation of active heterodimeric luciferase.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.     

Cloning and sequence analysis of a cDNA for human glycosylasparaginase. A single gene encodes the subunits of this lysosomal amidase

We have isolated a full-length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221-bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the alpha and beta subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post-translationally processed to generate two subunits of approx. 19.5 (alpha) and 15 (beta) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the alpha and beta subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.     

Cloning and expression of a divergent integrin subunit beta 8

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.     

The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.     

N-terminal amino acid sequence analyses of the alpha and beta polypeptides from four distinct subsets of sheep major histocompatibility complex class II molecules

Four monoclonal antibodies, SBU.II 28-1, 37-68, 38-27, and 42-20, each recognizing a distinct, non-overlapping subset of sheep class II molecules, were used to purify class II molecules from a single sheep. Four class II alpha subunits designated 28-1 alpha, 37-68 alpha, 42-20 alpha, and 38-27 alpha and five class II beta subunits designated 28-1 beta, 37-68 beta 1, 37-68 beta 2, 42-20 beta, and 38-27 beta were compared by N-terminal sequence analyses. Two distinct alpha subunits were identified; the 28-1 alpha, 37-68 alpha, and 42-20 alpha subunits all had identical N-terminal amino acids sequences, which exhibited about 75% homology with HLA-DR alpha and mouse E alpha polypeptides. In contrast, the 38-27 alpha sequence exhibited about 80% sequence homology with HLA-DQ alpha and mouse A alpha polypeptides. In general, sheep beta subunits displayed insufficient sequence homology to enable correlation with human beta-chain sequences; however, the 38-27 beta-chain sequence showed homology with the HLA-DQ beta sequence. The conserved sequence surrounding the site for N-linked glycosylation within human/mouse beta polypeptides (residues 19 to 21) was not present in sheep beta sequences and in contrast with the beta-chains of mouse and man, sheep beta polypeptides contained between 1 and 3 positionally variable cysteine residues (residues 13 to 15 inclusive). Individual sheep beta subunits exhibited extensive sequence heterogeneity and each consisted of a unique population of beta polypeptide species. At least 16 different beta polypeptide sequences were identified from a single sheep and the existence of no fewer than nine non-allelic beta genes was inferred from the sequence data. We have previously provided evidence suggesting that the sheep has multiple major histocompatibility complex class II alpha and beta genes related to those of all three HLA-D subregions. The present results suggest that a number of these genes encode HLA-DQ-like heterodimers and that a sheep DR-like alpha gene product is shared with the products of a large and heterogeneous sheep beta gene family.     

Sequence and structural analysis of the alpha- and beta-dinitrogenase subunits of Thiobacillus ferrooxidans

The structural genes (nifD and nifK) for the alpha and beta subunits of the molybdenum-iron (MoFe) protein of the Thiobacillus ferrooxidans dinitrogenase have been sequenced. The Mr values deduced from the nucleotide sequences are 54,919 and 57,901 for the alpha and beta subunits, respectively. The amino acid sequences of both subunits were quantitatively compared with the equivalent subunits from other bacteria. Distinct areas of amino acid homology were found between the alpha and beta subunits of T. ferrooxidans.