Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

Selective AmpliGfi atLon of DNA Fragments Coding for the n of Factors IF2 and EF-Tu, Two G Proteins from the Myxobacterium aurantiaca

L. Bremaud, B. Derijard, 1 and Y. Cenatiempo

Institut de Biologie Mol~culaire et d'Ing~nierie G6n~tique, Centre National de la Recherche Scientifique (CNRS) URA1172, Universit~ de Poitiers, 86022 Poitiers Cedex, France

Two DNA fragments of the genome of Selective amplification of DNA frag- bacterial species, because under starva- the myxobacterlum $tigmotella au- ments is desired as the first step in gene tion these prokaryotic cells undergo a rantlaca were selectively amplified by cloning experiments, especially when developmental cycle leading to the for- PCR. These fragments encode a seg- the use of homologous probe is required. mation of particular structures termed ment of the G domain of transla- This situation occurs when dealing with fruiting bodies. (7'8) Multicellular devel- tional initiation factor IF2 and elon- a family of proteins with members that opment of is sustained by gation factor EF-Tu, two GTP-binding display strong similarities in their pri- signal exchanges presumably transduced proteins. This was made possible by mary structure. In this respect, an inter- by G proteins, as in most eukaryotes. (9'1~ carefully designing the primers for esting example is provided by the widely Two soluble G proteins, translational ini- this reaction to avoid the amplifica- spread GTP-binding proteins (G pro- tiation IF2 and elongation factor EF-Tu, tion of every G-domain-encoding re- teins). may also play a role, directly or indi- gion of the genome. The sequence of The members of the G protein super- rectly, in this developmental process, via two pairs of primers was deduced family (1) are involved in diverse biolog- the selection and translation of particu- from highly conserved regions, ical processes such as transmembrane lar mRNAs. namely G1 and G 3 and/or their viol- signaling, control of proliferation and IF2 and EF-Tu are present in different hal amino acids, within each subfam- differentiation, and initiation and elon- forms in , and they arise from ily (initiation and elongation factors, gation of protein synthesis in prokary- different processes. In Escherichia coli, respectively) of GTP-binding pro- otes and eukaryotes. (z--s) In spite of these one gene, infg, directs the synthesis of teins. On the basis of the expected different roles, similar molecular mech- the various forms of IF2, caused by the size, one band was selected in each anisms occur for GTP binding (active presence of several translational initia- experiment, cloned into a vector, and form) followed by its hydrolysis into tion sites on infB mRNA, (11) whereas EF- sequenced. This showed unambigu- GDP (inactive form). Conserved primary Tu appears as two polypeptides bearing a ously after comparison analysis that and presumably tertiary protein struc- different amino acid at the carboxyl ter- they belong to the IF2 and EF-Tu tures were identified, especially within minus. The latter are coded by two genes, respectively. This strategy the G domain bearing the most con- genes, tufA and tufB, differing only in 13 seems suitable for the amplification served motifs. (L6) positions out of a total of 1185 nucle- of a segment of any gene coding for a It is now accepted that at least four otides; 8 of the 13 positions are located G protein from any origin. conserved regions, termed G~ to G4, are within the region encoding the G do- found throughout all the superfamily, main.(12,13) but with an even higher homology In this report we show that it is pos- within subgroups such as translational sible to amplify specifically a segment of initiation factors, elongation factors, the genes for IF2 and for EF-Tu. This was small eukaryotic GTPases like Ras pro- performed by designing different pairs teins, and subunits of signal-transducing of primers, taking into account the most G proteins. Consensus sequence motifs conserved sequences, but also the main have been proposed: GXXXXGKS#I', differences among subfamilies, within D-Xn-T, DXXG, and NKXD within G~, and at the border of the consensus re- 1Present address: Howard Hughes Medical Institute G2, G3, and G4, respectively. (~) gions G~ and G3. Research Laboratories, University of Massachusetts Medical Center, II Biotech Park, Worcester, Massachu- We began studying GTP-binding pro- The amplified DNA fragments, which setts 01605 USA. teins in Stigmatella aurantiaca, a myxo- were identified unambiguously by se-

3:195-1999 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $5.00 PCR Methods and Applications 195 Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

quencing, will now be used as homolo- appended with EcoRI and BamHI restric- fication with Tu primers (tuf G1 and gous probes to clone the corresponding tion sites at their 5' terminus. G'3a ) was almost identical, except that genes of S. aurantiaca. the annealing temperature was dropped to 56~ PCR Amplification Twenty microliters of each reaction MATERIALS AND METHODS PCR was performed with a Gene ATAQ mixture was loaded onto a 2% agarose Bacterial Strains and Pharmacia Thermocycler. Amplification gel. After electrophoresis, amplification Growth Conditions of the desired segments was performed products were visualized by ethidium in a 50-p4 reaction mixture containing 1 bromide staining. PCR products were S. aurantiaca DW4 was obtained from D. ~g of S. aurantiaca genomic DNA; 50 purified using the freeze-squeeze White (Indiana University, Blooming- pmoles of each primer; 200 p.M each of method. (~7) After extraction and ethanol ton). This strain was grown at 30~ in 1% dATP, dTTP, dCTP, and dGTP; Taq DNA precipitation, amplification products Bactocasitone (Difco), with 8 mM polymerase buffer [10 mM Tris-HCl (pH were digested with BamHI (BRL) and MgSO4, and harvested at -4.108 cells/ 9), 50 mM KC1, 1.5 mM MgCl2, 0.1% Tri- EcoRI (BRL). DNA fragments were sub- ml. E. coli TG1 was grown at 37~ in 2x ton X-100; and 1.25 units of Taq DNA cloned into M13mp19, digested previ- YT medium. (~4) polymerase. ously with the same restriction enzymes, Phenol-extracted S. aurantiaca ge- and transformed into E. coli TG1. The DNA Preparation nomic DNA was denatured for 5 min at single-stranded template was sequenced 100~ in the presence of both primers, by the dideoxy chain termination Genomic DNA of S. aurantiaca DW4 was overlaid with 50 ~1 of mineral oil method, (18) using a fluorescent universal extracted by the procedure described by (Sigma), and then cooled rapidly on ice. M13 primer and T7 DNA polymerase. Se- Starich and Zissler. (~s) Next, dNTPs, Taq DNA polymerase quencing was performed with an auto- buffer, water, and Taq DNA polymerase mated sequencer (A.L.F., Pharmacia). were added to the mineral oil and Primer Synthesis dropped by a brief centrifugation. Degenerate oligonucleotides used for Amplification with IF2 primers (infB Computer Analysis of PCR were produced by the phosphora- G1 and G3a) was carried out as follows: DNA Sequences midite method as described previ- Each cycle consisted of denaturation at Computer analysis of PCR fragments was ously, (16) using a Gene Assembler Syn- 94~ for 30 sec, annealing at 65~ for 30 performed using DNAsis software (Hita- thesizer (Pharmacia LKB). Upstream and sec, and extension at 72~ for 1 rain. chi). downstream primers, respectively, were This cycle was repeated 30 times. Ampli-

RESULTS Primer Design The first stage of this work was to design G 1 G 3 two sets of primers to amplify specifi- GHVDHGK, .DxPG ...... VVTIMGH ...... ITFLDTP ...... cally a segment of the genes infB and tuf of S. aurantiaca coding for two GTP- I 162 bp binding proteins, repectively, IF2 and ...... NIGTIGHV ...... ~ ..... NMITGAAQ .... EF-Tu. As shown in Figure 1A, to obtain --tuf G-~, <--tuf G~- i 255 bp an amplification of a G-protein-encod- ing gene segment, we took advantage of (1) conserved amino acid sequences G 1

infB G1 5' CGGAATTC GTG GTG ACC ATC ATG GG~ CAC 3" and G3 to design the primers inf8 G1, C C G T infB G3a, and tufG 1, which overlap those consensus sequences and upstream tuf GI 5' CGGAATTC AAC ATC GGC ACG ATC GGC CAC GTT 3' C amino acids; and (2) a conserved stretch of 8 amino acids, located downstream infB G3a 5' GCGGATCC GGG GGT GTC ~AG GAA GTT GAT 3' c c ~ G C from G3, in the Tu subgroup of the G-protein family to design tuf G'3a. tuf G'3a 5' GCGGATCC CTG ~GC ~GC GCC ~GT GAT CAT GTT 3' G G G A We had to take into account the GC content (-70%) of the myxobacterial ge- nome, especially at the third position of FIGURE 1 Conserved amino acid sequences in the G domain of IF2 and EF-Tu (A) and nucleotide codons where it can be as high as sequence of primers used in PCR (B). (A) G1 and G 3 represent two consensus motifs found 90%. (19) Moreover, the high GC content throughout G proteins; broken lines correspond to IF2 and EF-Tu, respectively. Only those amino of the primers could theoretically lead to acids taken into account to design the primers (infB G1, infB G 3, tuf G1, and tuf G'3a) are dis- played. The size (in bp) of the expected PCR fragments is also indicated. (B) The nucleotide secondary structures, because of self-an- sequence of degenerate primers is described. At degenerated positions, in the absence of any nealing, and to primer-dimer forma- other indication, each base represents 50% of the mixture, whereas underlined or doubly un- tion. This was estimated by using the derlined bases represent 66.6% and 83.3%, respectively, of the mixture. OLIGOTEST version 2.0 software, (2~

196 PCR Methods and Applications Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

the IF2 gene. A major amplified product Identification of PCR Fragments of the same size was obtained with E. coli and S. aurantiaca DNA (lanes 2 and 3). The putative infB and tuf segments of S. Two weaker bands of a smaller size were aurantiaca were inserted into M13mp19 observed with the latter. The lower of before sequencing. A comparison of the these two bands may correspond to an DNA sequence of these PCR fragments amplification product arising from infB (excluding the above described ap- G3a alone (lane 5), whereas the origin of pended restriction sites) with their E. coli the other one remains unexplained. The counterpart is presented in Figure 3. The expected size of the major band was 178 size of the corresponding fragments is FIGURE 2 Agarose gel analysis of the PCR-am- bp (162 bp plus 16 bp attributable to the absolutely identical in both bacterial plified products. (Left) The size of relevant appended EcoRI and BamHI sites). This species. A 71.5% identity was calculated markers separated in lane 1 (pBluescript re- length is compatible with its observed between E. coli and S. aurantiaca 162-bp stricted with HpalI); (lanes 2,3) amplification size (180--200 bp). fragments (Fig. 3, bottom), leading to a with infB G 1 and infB G3a from E. coli and S. In the case of the second set of prim- 81.5% amino acid identity (Fig. 4, bot- aurantiaca genomic DNA, respectively; (lanes ers, a unique band seems to correspond tom). Therefore, one can conclude that 4-6) control experiments with infB G1 alone, to an amplified segment of the E. coli the S. aurantiaca fragment has been am- infB G3a alone, and without DNA, respec- tively; (lanes 7,8) amplification similar to EF-Tu gene (tufA or tufB, lane 7), whereas plified from the infB gene. The situation lanes 2 and 3 but with tuf G~ and tuf G'3a; the corresponding band amplified from may be more complex with the 255-bp (lanes 9,10) controls with tufG1 alone and tuf the genome of S. aurantiaca appears fragment, as two genes for EF-Tu may be G'~, respectively. (Right) The positions of the weaker than a shorter one, presumably a present within the genome of S. auranti- expected bands are indicated by arrows and primer-dimer or a nonspecific amplified aca. However, there is little doubt about their theoretical sizes are shown. product (lane 8). However, we did not the nature of this fragment, which seems try to optimize further the PCR, as the to belong to a tufgene. When compared upper band migrates at the expected with the E. coli tufB sequence (Fig. 3, level (-270 bp) and the estimated top), the percentage of identity reaches which indicated that self complementa- amount of DNA was sufficient for subse- 69.4% (76.5% for the deduced protein se- rity within the primers did not exceed 5 quent cloning. quence; Fig. 4, top). Whether the se- bp but that contiguous and noncontigu- ous complementarity could occur be- tween mainly tuf G1 and tuf G'3~, thus leading to primer-dimer formation in S. aurantiaca AACATCGGCACGATCGGCCACGTTGACCACGGGAAGACGTCGCTGACGGCCGCCATCACC PCR. Finally, directional cloning in a se- ...... quencing vector was done by adding E. coli AACGTCGGTACTATCGGCCACGTTGACCATGGTAAAACAACGCTGACCGCTGCAATCACT 770 780 790 800 810 bases at the 5' terminus of the primers (restriction sites). S. aurantiaca AAGGTGCTGGCCAAGACGGGCGGCGCCACGTTCCTGGCCTATGACAGAATCGACAAGGCC E. coli ICC~A~TiiAi~CTA~GTG~TGCT~GC~A+TC~I~CAGi+~iT~C~G Figure 1B shows the sequence of the 83o 84o 850 86o 87o degenerate primers that were synthe- S. aurantiaca CCCGAGGAGCGTGAGCGCGGCATCACCATCTCCACGGCGCACGTGGAGTACCAGACGAAG sized following the designed specifica- E. coli ~G~iA~AAAAA~CT~T~T~t~it~AA~A~TT~T~T~AA~A~GACA~CCC~ tions. As indicated, only a few positions 890 900 910 920 930 were degenerated on the basis of codon S, aurantiaca AACCGCCACTACGCGCACGTGGACAGTCCTGGCCACGCCGACTACGTGAAGAACATGATT ...... bias determined from the analysis of 18 ...... E. coli ACCCGTCACTACGCACACGTAGACTGCCCGGGGCACGCCGACTATGTTAAAAACATGATC sequenced genes of another myxobacte- 950 960 970 980 990 rium, (B. D~rijard, S. aurantiaca ACCGGCGCGGCGCAG unpubl.). This bacterial species was cho- E. coli ACCGGTGCTGCGCAG sen because insufficient data were avail- i010 able to define codon preferences in the genome of S. aurantiaca. S. aurantiaca GTGGTGACGATCATGGGCCACGTCGACCACGGCAAGACGAGCCTCCTGGACGCCATCCGC ...... E. coli GTTGTGACCATCATGGGTCACGTTGACCACGGTAAAACCTCTCTGCTGGACTACATTCGT Amplification 1330 1340 1350 1360 1370 S. aurantiaca GCGGCCAACGTGGCCTCGGGTGAGGCCGGCGGCATCACCCAGCACATCGGCGCCTACAGC PCR experiments were performed with E. coli T~AACGIIAGTGGCCTCTG~C~iA~GGGCGGCATTA~AGCICA#T~T~A+A~CA~ the two pairs of primers, infB G1/infB 1390 1400 1410 1420 1430 G3a, and tuf G1/tuf G'~a, and included S. aurantiaca GTCGCCACGGCCCGGGGAGACATCAAGTTCCTCGACACCCCC negative and positive controls as dis- E, coli played in Figure 2. Positive controls were 1450 1460 1470 carried out by using E. coli genomic DNA, the IF2 and EF-TU G domains of FIGURE 3 Nucleotide sequence of the PCR fragments from S. aurantiaca and their alignment with which were already described. (2'B1'22) E. coli tufB (top) and infB (bottom) regions coding for part of the G domain of EF-Tu and IF2, The first set of primers, infB G~ and G3a, respectively. The nucleotide sequence of the restriction sites added to the 5' end of the primers was used to amplify a segment of infB, is not displayed (see text and Fig. 1). Identical bases are indicated by colons.

PCR Methods and Applications 197 Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

tween tuf genes have been described in E. coli E. coli, and were not able to come to a S. aurantiaca GT I GH VDHG KT LTAA I T conclusion because three types of situa- tions were encountered: At some posi- E. coli S. aurantiaca tions a nucleotide is identical to that present in tufA (such as a C at nucleotide E. coli S. aurantiaca 789), or in tufB (such as a C at nucleotide t i 800), or even absent in both genes (such E. coli PG ADY VKNM I T G as a G at nucleotide 798). Therefore, the S. aurantiaca PG~ADYVKNM I sequence of the entire gene as well as the surrounding region of the S. aurantiaca genome is required to solve this prob- lem, because tufA is part of a complex S. aurantiaca V T I D G S L L operon including a gene (fusA) coding for the EF-G, another G protein, whereas S. aurantiaca V S G E G G I O H I G A Y tufB is preceded by tRNA genes. (1z'13) Finally, an analysis of the deduced protein sequences shows a very high de- ,, gree of conservation within their G do- mains and confirms that a choice of FIGURE 4 Amino acid sequence of S. aurantiaca gene products deduced from Fig. 3 aligned with primers designed only from consensus a segment of EF-TU (top) and IF2 (bottom). Conserved sequences are boxed. sequences such as G 1 or G3 could have led to the simultaneous amplification of at least two G-domain-encoding regions. For instance, a long stretch of identical quenced fragment represents the equiv- position, 96% (infB) and 92% (tuf). This amino acids is found within and after G1 alent of tufA or tufB gene cannot be establishes that we are dealing with a of IF2 and EF-Tu of S. aurantiaca. assessed from this simple alignment (see DNA amplified from myxobacteria and Either amplified fragments or oligo- Discussion). rules out any possibility of amplification nucleotides generated from the most di- from contaminating DNA, especially E. verged regions within the sequenced coll. segments could be used to clone individ- DISCUSSION Finally, we compared these new se- ual genes. We chose the amplified frag- The goal of this work was to define a quences with homologous regions in the ments as homologous probes to screen a simple strategy to obtain a homologous genome of E. coli and other microorgan- hgtll library of S. aurantiaca DW4/3.1 probe to increase the probability of clon- isms. Four infB sequences have already genomic DNA (kindly provided by H.U. ing some, but not all, genes coding for G been published, out of which only one is Schairer, Zentrum fiir Molekular Biolo- proteins in a myxobacterial species, S. from Gram-negative bacteria (E. coli) (zl) gie, University of Heidelberg, Germany). aurantiaca. We thought that the choice and three from gram-positive ones (Ba- On the basis of preliminary sequence of primers based on consensus se- cillus subtilis, Streptococcus thermophilus, data, we have identified clones bearing quences within the G domain but lim- and Streptococcus faecium). (23-zs) It is in- infB and tufB, respectively (L. Bermaud, ited to subfamily homologies was criti- teresting, knowing that myxobacteria B. D6rijard, and Y. Cenatiempo, in cal. Furthermore, we expected the main are Gram-negative bacteria, that the ho- prep.). problem to be the nature of the myxo- mology found with the corresponding The strategy proposed here may be bacterial genome, namely its high GC sequences is the highest with the E. coli extended to other sets of primers and content. According to the nucleotide se- genome (71.5%). Lower percentages are eventually different prokaryotic or eu- quence of the amplified products, the obtained with the above mentioned karyotic organisms. This would allow for strategy appears to be successful in the Gram-positive bacteria (66.5%, 68%, and the cloning of any gene coding for a two chosen cases (infB and tuf seg- 60%, respectively). Such values extended member of the very important super- ments), in spite of the fact that the am- to the overall gene will be important in family of G proteins. plification was obscured in the second terms of evolutionary studies. example, probably by primer-dimer for- The presence of two tuf genes in E. ACKNOWLEDGMENTS mation. coli 0z'13), or even three in Streptomyces We are grateful to Didier Delourme for Although these segments represent ramocissimus (L.P. Woudt, K. Rietveld, M. his help in the preparation of this manu- relatively short portions of the genes, in- Verdurmen, J. Van Haarlem, G.P. Van script. teresting information can already be de- Wezel, E. Vijgenboom, and L. Basch, un- duced from their nucleotide sequences. publ.), raises the question of whether NOTE ADDED IN PROOF The calculated GC percent in the infB se- such a situation occurs in S. aurantiaca quence, including the primers, reaches and, consequently, which fragment has The nucleotide sequence data reported 75%, a value higher than that observed been amplified in our experiments. We in this paper will appear in the EMBL, in the tufsequence, -65%. More impres- examined the sequence, especially at GenBank, and DDBJ nucleotide se- sive is this percentage at the third codon those positions where differences be- quence data bases under the accession

198 PCR Methods and Applications Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

numbers X75300 and X75301 for tufB 14. Davis, L.G., M.D. Dibner, and J.F. Battey, and infB, respectively. eds. 1986. Basic methods in molecular biol- ogy. Elsevier, Amsterdam, The Netherlands. 15. Starich, T. and J. Zissler. 1989. Movement REFERENCES of multiple DNA units between Myxococ- 1. Bourne, H.R., D.A. Sanders, and F. McCor- cus xanthus cells. J. BacterioL 171: 2323- mick. 1991. The GTPase superfamily: 2336. Conserved structure and molecular mech- 16. Hartmann, R.K., N. Ulbrich, and V.A. Erd- anism. Nature 349: 117-127. mann. 1987. An unusual rRNA operon 2. Cenatiempo, Y., F. Deville, J. Dondon, M. constellation: In Thermus thermophilus Grunberg-Manago, C. Sacerdot, J.W.B. HB8 the 23S/5S rRNA operon is a separate Hershey, H.F Hansen, H.U. Petersen, entity from the 16S rRNA operon. Bio- B.F.C. Clark, M. Kjieldgaard, T.F.M. La chimie 69: 1097-1104. Cour, K.K. Mortensen, and J. Nyborg. 17. Tautz, D and M. Renz. 1983. An optimized 1987. The protein synthesis initiation fac- freeze-squeeze method for the recovery of tor 2 G-domain. Study of a functionally DNA fragments from agarose gels. Anal. active C-terminal 65-kilodalton fragment Biochem. 132: 14-19. of IF2 from Escherichia coll. Biochemistry 18. Sanger, F., S. Nicklen, and A.R. Coulson. 26: 5070-5076. 1977. DNA sequencing with chain termi- 3. Pfeuffer, T. and E.J.M. Helmreich. 1988. nation inhibitors. Proc. Natl. Acad. Sci. 74: Structural and functional relationships of 5463-5467. guanosine triphosphate binding proteins. 19. Mesbah, M., U. Premachandran, and W.B. Curr. Topics Cell Regul. 29: 129-216. Whitman. 1989. Precise measurement of 4. Bourne, H.R., D.A. Sanders, and F. McCor- the G+C content of deoxyribonucleic mick. 1990. The GTPase superfamily: A acid by high-performance liquid chroma- conserved switch for diverse cell func- tography. Int. ]. Syst. Bacteriol. 39: 159- tions. Nature 348: 125-132. 167. 5. Gibbs, J.B. and M.S. Marshall. 1989. The 20. Beroud, C., C. Antignac, C. Jeanpierre, ras oncogene--An important regulatory and C. Junien. 1990. Un programme in- element in lower eucaryotic organisms. formatique pour la recherche d'amorces Microbiol. Rev. 53: 171-185. pour l'amplification par PCR. M~decine/ 6. Dever, T.E., M.J. Glynias, and W.C. Mer- Sciences 9" 901-903. rick. 1987. GTP binding domain: Three 21. Sacerdot, C., P. Dessen, J.W.B. Hershey, J. consensus elements with distinct spacing. Plumbridge, and M. Grunberg-Manago. Proc. Natl. Acad. Sci. 84: 1814-1818. 1984. Sequence of initiation factor IF2 7. Wireman, J.W. and M. Dworkin. 1975. gene: Unusual protein features and ho- Morphogenesis and developmental inter- mologies with elongation factors. Proc. actions in myxobacteria. Science 189: Natl. Acad. Sci. 81: 7787-7791. 516-522. 22. Laalami, S., C. Sacerdot, G. Vachon, K. 8. Shimkets, L.J. 1990. Social and develop- Mortensen, H.U. Sperling-Petersen, Y. mental biology of the myxobacteria. Mi- Cenatiempo, and M. Grunberg-Manago. crobiol. Rev. 54: 473-501. 1991. Structural and functional domains 9. D6rijard, B., M. Ben AYssa, B. Lubochin- of E. coli initiation factor IF2. Biochimie sky, and Y. Cenatiempo. 1989. Evidence 73: 1557-1566. for a membrane-associated GTP-binding 23. Brombach, M., C.O. Gualerzi, Y. Naka- protein in Stigmatella aurantiaca, a mura, and C.L. Pon. 1986. Molecular prokaryotic cell. Biochem. Biophys. Res. cloning and sequence of the Bacillus Commun. 158: 562-568. stearothermophilus translational initiation 10. March, P.E. 1992. Membrane-associated factor IF2 gene. Mol. Gen. Genet. 205: 97- GTPases in bacteria. Mol. Microbiol. 6: 102. 1253-1257. 24. Friedrich, K., M. Brombach, and C.L. Pon. 11. Sacerdot, C., G. Vachon, S. Laalami, F. 1988. Identification, cloning and se- Morel-Deville, Y. Cenatiempo, and M. quence of the Streptococcus faecium infB Grunberg-Manago. 1992. Both forms of (translational initiation factor IF2) gene. translational initiation factor IF2 (a and Mol. Gen. Genet. 214: 595-600. ~) are required for maximal growth of Es- 25. Shazand, K., J. Tucker, R. Chiang, K. cherichia coll. Evidence for two transla- Stansmore, H.U. Sperling-Petersen, M. tional initiation codons for IF2~. J. Mol. Grunberg-Manago, J.C. Rabinowitz, and Biol. 225: 67-80. T. Leighton. 1990. Isolation and molecu- 12. Yokota, T., H. Sugisaki, M. Takanami, and lar genetic characterization of the Bacillus Y. Kaziro. 1980. The nucleotide sequence subtilis gene (in,B) encoding protein syn- of the cloned tufA gene of Escherichia coll. thesis initiation factor 2. J. Bacteriol. Gene 12: 25-31. 172: 2675-2687. 13. An, G. and J.D. Friesen. 1980. The nucle- otide sequence of tufB and four nearby tRNA structural genes of Escherichia coli. Received August 23, 1993; accepted in Gene 12: 33-39. revised form October 14, 1993.

PCR Methods and Applications 199 Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

Selective amplification of DNA fragments coding for the G domain of factors IF2 and EF-Tu, two G proteins from the myxobacterium Stigmatella aurantiaca.

L Bremaud, B Derijard and Y Cenatiempo

Genome Res. 1993 3: 195-199

References This article cites 24 articles, 8 of which can be accessed free at: http://genome.cshlp.org/content/3/3/195.full.html#ref-list-1

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

To subscribe to Genome Research go to: https://genome.cshlp.org/subscriptions

Copyright © Cold Spring Harbor Laboratory Press