Sequence analysis and expression of the two genes for elongation factor 1 alpha from the dimorphic yeast Candida albicans.

Two Candida albicans genes that encode the protein synthesis factor elongation factor 1 alpha (EF-1 alpha) were cloned by using a heterologous TEF1 probe from Mucor racemosus to screen libraries of C. albicans genomic DNA. Sequence analysis of the two clones showed that regions of DNA flanking the coding regions of the two genes were not homologous, verifying the presence of two genes, called TEF1 and TEF2, for EF-1 alpha in C. albicans. The coding regions of TEF1 and TEF2 differed by only five nucleotides and encoded identical EF-1 alpha proteins of 458 amino acids. Both genes were transcribed into mRNA in vivo, as shown by hybridization of oligonucleotide probes, which bound specifically to the 3' nontranslated regions of TEF1 and TEF2, respectively, to C. albicans total RNA in Northern (RNA) blot analysis. The predicted EF-1 alpha protein of C. albicans was more similar to Saccharomyces cerevisiae EF-1 alpha than to M. racemosus EF-1 alpha. Furthermore, codon bias and the promoter and termination signals of the C. albicans EF-1 alpha proteins were remarkably similar to those of S. cerevisiae EF-1 alpha. Taken together, these results suggest that C. albicans is more closely related to the ascomycete S. cerevisiae than to the zygomycete M. racemosus.     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

Identification of two genes coding for the translation elongation factor EF-1 alpha of S. cerevisiae.

The translation elongation factor EF-1 alpha of the yeast Saccharomyces cerevisiae is coded for by two genes, called TEF1 and TEF2. Both genes were cloned. TEF1 maps on chromosome II close to LYS2. The location of TEF2 is unknown. TEF2 alone is sufficient to promote growth of the cells as shown with a strain deleted for TEF1. TEF1 and TEF2 were originally identified as two strongly transcribed genes, which most likely code for an identical or nearly identical protein as judged from S1 nuclease protection experiments with mRNA-DNA hybrids. The DNA sequence analysis of TEF1 allowed the prediction of the protein sequence. This was shown, by a search in the Dayhoff protein data bank, to represent the translation elongation factor EF-1 alpha due to the striking similarity to EF-1 alpha from the shrimp Artemia. A search for TEF1 homologous sequences in several yeast species shows, in most cases, duplicated genes and a much higher sequence conservation than among genes encoding amino acid biosynthetic enzymes.     

Identification of nitrogenase and carboxylase genes in the photosynthetic bacteria and cloning of a carboxylase gene from Rhodopseudomonas sphaeroides

The presumptive genes for the ribulose 1,5-bisphosphate carboxylase large subunit and for nitrogenase-specific components from Rhodopseudomonas sphaeroides and several other photosynthetic bacteria were identified and located by interspecific probing. Restriction digests of R. sphaeroides genomic DNA were hybridized under stringent conditions to cloned DNA from Rhodospirillum rubrum (plasmid pRR2119 carrying the carboxylase gene) and Klebsiella pneumoniae (pSA30 carrying the nitrogenase genes). The nitrogenase probe hybridized with different signal intensities to several distinct HindIII, BglII, EcoRI, BamHI and PvuII fragments of R. sphaeroides 2.4.1.DNA. The carboxylase probe hybridized to only single R. sphaeroides 2.4.1.DNA fragments produced with all five restriction enzymes. A 3000-bp EcoRI-BamHI R. sphaeroides 2.4.1.DNA fragment carrying the presumptive gene for the large subunit of ribulose 1,5-bisphosphate carboxylase was cloned into pBR322 and positively identified by probing with a 32P-labeled internal PstI fragment of the Rhodospirillum carboxylase gene.     

The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.     

Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.     

Polymorphic class II sequences linked to the rat major histocompatibility complex (RT1) homologous to human DR and DQ sequences

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences.     

The ornithine aminotransferase-encoding gene family of rat: cloning, characterization, and evolutionary relationships between a single expressed gene and three pseudogenes

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.     

Apis mellifera cytoplasmic elongation factor 1 alpha (EF-1 alpha) is closely related to Drosophila melanogaster EF-1 alpha

Using low stringency hybridisation with a Drosophila melanogaster EF-1 alpha gene fragment we have isolated a genomic DNA clone encoding elongation factor 1 alpha (EF-1 alpha) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF-1 alpha sequences further confirms that certain amino acid sequences seem to be invariable within the EF-1 alpha protein family.     

A general upstream binding factor for genes of the yeast translational apparatus.

Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

At least two separate gene clusters are involved in albicidin production by Xanthomonas albilineans.

Transposon mutagenesis was used to obtain mutations affecting production of the toxin albicidin in Xanthomonas albilineans, which causes leaf scald disease of sugarcane and is also pathogenic to corn. Transposon Tn5-gusA inserted randomly into genomic DNA of X. albilineans Xa23R1 at a frequency of 10(-4) to 10(-5) per recipient after conjugal transfer from Escherichia coli. Fifty prototrophic mutants defective in albicidin production were isolated from 7,100 Tn5-gusA insertional derivatives tested for toxin production by an antibiosis bioassay. EcoRI fragments containing Tn5 flanking sequences from two mutants (AM15 and AM40) were cloned and used to probe a wild-type Xa23R1 DNA library by colony hybridization. Nine cosmids showed homology to the AM15 probe, and six showed homology to the AM40 probe. Four cosmid clones hybridized to both probes. Forty-five of the 50 defective mutants were restored to albicidin production with two overlapping cosmid clones. Restriction mapping showed that these mutants span a genomic region of about 48 kb. At least one other gene cluster is also involved in albicidin production in Xa23R1. DNA fragments from the 48-kb cluster proved to be very specific to X. albilineans. Some mutants affected in albicidin production retain their ability to colonize sugarcane cultivated in vitro.     

Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E1 alpha; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a lambda gt11 cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E1 alpha. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E1 alpha. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E1 alpha and the 5' end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids (Mr = 42,703). Hybridization analysis showed that the size of the mRNA is about 1.4 kilobases.     

Map position and genomic organization of the kps cluster for polysialic acid synthesis in Escherichia coli K1.

The multigenic kps cluster in Escherichia coli K1 encodes functions for synthesis of a polysialic acid capsule. DNA probes flanking each side of the cluster were hybridized to lambda clones bearing overlapping E. coli W3110 genomic fragments. These fragments covered the region between 60 and 70 map units on the chromosome. The results located kps to an accretion domain near 64 map units and established the orientation of kps cluster genes. Acquisition of kps by the E. coli genome was apparently the result of an ancestral transpositionlike addition event.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.