Patent Application: US-63239803-A

Abstract:
the present invention provides materials and methods for introducing genetic disruptions in bacterial genetic material , especially in that of streptomyces spp .. a novel transposon is provided , which has an origin of transfer between inverted repeat sequences . the transposon may also include a genetic marker . the transposon introduces a disruption into dna of interest , which disruption may then be conjugated into host bacteria , including bacteria of other species or strains . the host bacteria is incubated at conditions suitable for homologous recombination between the conjugated dna and the host dna . the effect of the disruption in different genetic backgrounds can therefore be investigated . the disruption may be stored as a mobile genetic element ready for transfer to a test host .

Description:
a procedure for efficient systematic mutagenesis of streptomycete genes is described . the technique employs in vitro transposon mutagenesis , using a novel transposon tn5062 . mutations are initially derived in cloned streptomycete dna propagated in escherichia coli . the mutations are then mobilised into a streptomycete host in which marker replacement by homologous recombination occurs . the incorporation of a promoter - less copy of the egfp reporter gene in tn5062 permits temporal and spatial analysis of expression of a transposon tagged gene . [ 0075 ] streptomyces coelicolor m145 was used as a test strain for transposition and was cultured on sfm agar using standard techniques according to kieser et al . ( 2000 ). all dna manipulations were carried out using escherichia coli jm109 ( yanisch - perron et al ., 1985 ) or escherichia coli sure ( stratagene ). e . coli was grown on either l agar plates , l broth or 2xyt broth ( sambrook et al . 1989 ). e . coli et12567 ( macneil et al ., 1992 ) carrying puz8002 ( kieser et al ., 2000 ) was used as a host to mobilize transposed cosmids into s . coelicolor m145 . oligonucleotides and plasmids / cosmids used in this study are listed in tables 1 and 2 respectively . plasmid isolations were carried out using wizard sv kits from promega and cloning steps were performed using established procedures . restriction endonucleases and t4 ligase ( obtained from new england biolabs , life technologies or promega ) were used according to the manufacturer &# 39 ; s instructions . the first step in the construction of the transposon , tn5062 ( fig1 ), was to clone egfp from pegfp - n1 to palter1 as a 787 bp hindiii - xbai fragment creating pfp11 ( fig2 ). this allowed an ndei site to be introduced at the start codon of egfp by site directed mutagenesis using the altered sites kit from promega according to the manufacturer &# 39 ; s instructions resulting in pfp12 ( accatg ( pfp11 ) was changed to catatg ( pfp12 ); where atg is the first codon of the egfp gene ). the three frame translational stop was constructed as a linker made from the oligonucleotides vc1 and vc2 ( mwg - biotech ) and cloned into bglii - ndei digested pet26b + creating pvc101 . this was digested with ndei and xhoi and ligated to a second linker synthesised as the oligonucleotides vc3 and vc4 carrying a streptomyces consensus ribosome binding site creating pvc102 . egfp was cloned from pfp12 into pvc102 as 725 bp ndei - eagi fragment giving pvc107 . aac3 ( iv ) was first moved to palter1 from php45ωaac as a 1783 bp ecori fragment creating pqm501 . this plasmid was digested with smai and the 1794 bp aac3 ( iv ) fragment introduced between the tn5 inverted repeats of pmod & lt ; mcs & gt ; by blunt - ended ligation with ecoicri and hincii digested pmod & lt ; mcs & gt ; resulting in pqm504 . orit was introduced into pqm504 as a 786 bp psti fragment from pij8660 giving pqm5052 . finally egfp was added to pqm5052 as a 782 bp ecori fragment from pvc107 . this plasmid was named pqm5062 and allows tn5062 to be liberated from the plasmid backbone by digestion with pvuii as a 3442 bp fragment ( fig1 ). the sequence of the transposon was verified by restriction digestion with appropriate enzymes and sequencing using a beckman - coulter ceq 2000xl sequencer according to the manufacturer &# 39 ; s instructions . selected ( table 2 ) cosmids from the s . coelicolor a3 ( 2 ) cosmid library ( redenbach et al ., 1996 ) were obtained from helen kieser ( john innes centre , norwich , uk ) as e . coli sure cultures . cosmid dna was isolated from e . coli sure using wizard sv minipreps ( promega ) according to the manufacturer &# 39 ; s instructions . cultures were grown at 37 ° c . in l broth containing ampicillin ( 50 μg / ml ) and kanamycin ( 25 μg / ml ) and isolated dna transformed into e . coli jm109 by electroporation using a biorad gene pulser according to the manufacturer &# 39 ; s instructions on l agar plates containing ampicillin ( 50 μg / ml ) and kanamycin ( 25 μg / ml ). cosmid dna was again isolated using wizard sv kits according to the manufactures instructions except that cultures were incubated in 2 × yt broth containing ampicillin ( 50 μg / ml ) and kanamycin ( 25 μg / ml ) for exactly 18 hours at 250 rpm . dna was eluted from the spin column twice with 40 μl of 10 mm tris - hcl , ph 8 . 5 preheated to 50 ° c ., quantified spectrophotometrically ( od 260 ) using a beckman du 650 spectrophotometer and stored at − 70 ° c . pqm5062 dna was isolated using the wizard sv minipreps ( promega ) according to the manufacturer &# 39 ; s instructions after growth in l broth containing ampicillin ( 50 μg / ml ) and apramycin ( 100 μg / ml ). tn5062 was liberated from the plasmid by digestion with pvuii and electrophoresis on a 1 % agarose gel made with tae buffer . following electrophoresis the 3442 bp tn5062 band was excised from the gel using a scalpel and purified using the qiaex ii gel extraction system ( qiagen ) according to manufacturer &# 39 ; s instructions . following purification , tn5062 was further purified using the qiaquick pcr purification kit ( qiagen ) and ethanol precipitated before being resuspended in 10 μl of sterile distilled water and quantified by comparison with known standards after agarose gel electrophoresis . finally dna was stored at − 70 ° c . transposition of tn5062 into selected cosmids ( table 2 ) from the s . coelicolor cosmid library ( redenbach , et al ., 1996 ) was carried out by preparing the following reaction mix , in the listed order , in an eppendorf tube according to the manufacturer &# 39 ; s instructions ( epicentre ) and incubated for 2 hours at 37 ° c . : ez :: tn 10x reaction buffer 1 μl s . coelicolor a3 ( 2 ) cosmid dna 2 . 9 μl ( 200 ng or 7 . 6 × 10 − 9 μmoles ) tn5062 dna 1 . 2 μl ( 17 . 5 ng or 7 . 6 × 10 − 9 μmoles ) sterile distilled water 3 . 9 μl ez :: tn transposase 1 μl after completion , the reaction was stopped by adding 1 μl of ez :: tn stop solution and incubated at 70 ° c . for 10 minutes . 1 μl of the transposition reaction was then added to 40 μl of electrocompetent e . coli jm109 cells ( prepared according to manufacturer &# 39 ; s instructions ) and electroporated using a biorad gene pulser according to the manufacturer &# 39 ; s instructions . following electroporation cells were plated on l agar supplemented with ampicillin ( 50 μg / ml ), kanamycin ( 25 μg / ml ) and apramycin ( 100 μg / ml ) to select for colonies containing transposed cosmids . 96 ampicillin , kanamycin and apramycin resistant colonies were picked and inoculated to a 96 square well growth block ( abgene ), each well containing 1 ml of l broth containing ampicillin ( 50 μg / ml ), kanamycin ( 25 μg / ml ) and apramycin ( 100 μg / ml ). the block was then incubated overnight at 37 ° c ., 225 rpm . the next day 1 . 3 μl of each of the 96 cultures was the transferred to a second growth block , each well containing 1 . 3 ml of 2 × yt supplemented with apramycin ( 1000 g / ml ) and incubated for 18 hours at 37 ° c ., 225 rpm . 330 μl of 60 % ( w / v ) glycerol was then added to each of the 96 wells from the first growth block , mixed and stored at − 70 ° c . cosmid dna was isolated from the cultures in the second growth block using the wizard sv 96 kit from promega and stored at − 70 ° c . transposed cosmid dna ( 1 μl ) was first electrophoresed on a 0 . 7 ( w / v ) agarose gel to check dna quality . for sequencing 10 μl from each of the 96 samples was transferred to a 96 well pcr plate ( abgene ) and heated to 86 ° c . for 5 minutes in a mj research ptc - 200 dna engine . to each sample was then added 2 μl of transposon specific sequencing primer ezr1 ( 10 pmol / μl ) ( table1 , fig1 ) and 8 μl of ceq dtcs quick start sequencing kit ( beckman - coulter ). the sequencing reactions were then carried out in a mj research ptc - 200 dna engine by heating to 96 ° c . ( 20 seconds ), 55 ° c . ( 20 seconds ) and 60 ° c . ( 4 minutes ) for 50 cycles . the samples were then analysed on a ceq 200xl sequencer ( beckman - coulter ) using the long fast read program according to the manufacturer &# 39 ; s conditions . following sequencing the transposition target site was determined by comparison of each of the 96 sequences with the s . coelicolor a3 ( 2 ) genome sequence at http :// www . sanger . ac . uk / projects / s coelicolor /( bentley et al . 2002 ). identified insertions in cosmid sc7c7 are shown in table 3 . replacement of a wild type gene with the cosmid - borne transposed copy was carried out by conjugation from e . coli according to kieser et al . ( 2000 ). e . coli et12567 ( puz8002 ) was grown in the presence of kanamycin ( 25 μg / ml ) and chloramphenicol ( 25 μg / ml ) and chemically competent cells prepared according to sambrook et al . ( 1989 ). selected transposed cosmids were then transformed into these cells ( sambrook et al ., 1989 ) and grown on l agar supplemented with ampicillin ( 50 μg / ml ), kanamycin ( 25 μg / ml ) and apramycin ( 100 μg / ml ) to select for colonies containing transposed cosmids . the next day a single transformant was inoculated into 10 ml of l broth containing apramycin ( 100 μg / ml ), kanamycin ( 25 μg / ml ) and chloramphenicol ( 25 μg / ml ) and grown overnight at 37 ° c ., 250 rpm . the next day 0 . 4 ml of the overnight culture was added to 39 . 6 ml of l broth supplemented with apramycin ( 100 μg / ml ), kanamycin ( 25 μg / ml ) and chloramphenicol ( 25 μg / ml ), grown at 37 ° c . to an optical density of od 600 0 . 4 - 0 . 6 . at this point , cells were harvested by centrifugation and washed twice in 40 ml of l broth , before being resuspended in 4 ml of l broth . meanwhile , approximately 1 × 10 8 of s . coelicolor m145 spores were added to 500 μl of 2 × yt , heat shocked ( 50 ° c ., 10 minutes ) and allowed to cool . to this 500 μl of pregerminated spores was added 500 μl of the e . coli cells containing the transposed cosmid , after mixing , the cells and spores were centrifuged , most of the supernatant fraction removed and the pellet resuspended in the residual liquid . this was then plated on sfm agar , supplemented with 10 mm mgcl 2 and incubated at 30 ° c . for 16 hours . the next day the plate was overlayed with 1 ml of sterile distilled water supplemented with 1 mg of apramycin and 0 . 5 mg nalidixic acid and incubated at 30 ° c . for a further 3 - 4 days . after this time , individual transconjugants were picked off and patched onto sfm agar supplemented with nalidixic acid ( 25 μg / ml ) and apramycin ( 100 μg / ml ), similarly colonies were also patched onto sfm agar supplemented with nalidixic acid ( 25 μg / ml ) and kanamycin ( 25 μg / ml ). those colonies that had undergone a gene replacement and replaced the wild type gene with the cosmid borne copy containing the insertion sequence were identified on the basis of apramycin resistance and kanamycin sensitivity . sterile coverslips were inserted into sfm agar at a 45 ° angle and 10 μl of s . coelicolor a3 ( 2 ) spores were inoculated in the acute angle between coverslip and agar surface . after incubation for 1 - 7 days , coverslips were removed and washed twice by brief immersion in methanol . after drying , the coverslips were mounted on slides and examined microscopically using a nikon eclipse e600 fluorescence microscope . egfp expression was observed by illumination with ultra violet light and fluorescence visualised with a fitc filter set . although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding , it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims . for further information on techniques and materials , the addressee is referred also to general reference texts , such as sambrook et al ( 1989 ), kieser et al . ( 2000 ), ausubel et al . ( 1989 ), and any later editions thereof ( such as sambrook and russell ( 2001 ), as well as to the product literature of epicentre and other suppliers of commercially available transposition systems . each publication and earlier application referred to herein is hereby incorporated by reference in its entirety and for all purposes . [ 0098 ] table 2 plasmids and cosmids plasmid source pet26b + novagen palter1 promega pegfp - n1 clontech php45ωaac blondelet - rouault et al . ( 1997 ) pmod & lt ; mcs & gt ; epicentre pij8660 sun et al . ( 1999 ) pvc101 this work pvc102 this work pvc107 this work pqm501 this work pqm504 this work pqm5052 this work pqm5062 this work puz8002 kieser et al . ( 2000 ) sc1a6 redenbach et al . ( 1996 ) sc3a3 redenbach et al . ( 1996 ) sc6a9 redenbach et al . ( 1996 ) sc7b7 redenbach et al . ( 1996 ) sc7c7 redenbach et al . ( 1996 ) sce59 redenbach et al . ( 1996 ) scf91 redenbach et al . ( 1996 ) sch63 redenbach et al . ( 1996 ) sch69 redenbach et al . ( 1996 ) sci7 redenbach et al . ( 1996 ) sc4g10 redenbach et al . ( 1996 ) sc4b10 redenbach et al . ( 1996 ) sch66 redenbach et al . ( 1996 ) sc2e9 redenbach et al . ( 1996 ) sc9b5 redenbach et al . ( 1996 ) sci51 redenbach et al . ( 1996 ) 2sci34 redenbach et al . ( 1996 ) 2scg38 redenbach et al . ( 1996 ) scc88 redenbach et al . ( 1996 ) scc77 redenbach et al . ( 1996 ) sc9e12 redenbach et al . ( 1996 ) scf43 redenbach et al . ( 1996 ) sc5c11 redenbach et al . ( 1996 ) 2sck8 redenbach et al . ( 1996 ) sch44 redenbach et al . ( 1996 ) sc10f4 redenbach et al . ( 1996 ) scd66 redenbach et al . ( 1996 ) sce20 redenbach et al . ( 1996 ) scd16a redenbach et al . ( 1996 ) sch22a redenbach et al . ( 1996 ) scf55 redenbach et al . ( 1996 ) sc3c3 redenbach et al . ( 1996 ) sc2a11 redenbach et al . ( 1996 ) [ 0099 ] table 3 examples of transcriptional tn 5062 insertions in cosmid sc7c7 poten - tial egfp inser - tran - insertion insertion tion scrip - trans - position position strand tion target cosmid posant ( genome ) ( cosmid ) inserted orf (+/−) ( y / n /) site sc7c7 c12 6270990 295 13566 rrna 6269992 . . . 6271519 + rrne 16s + y cccttgtgg sc7c7 f04 271341 646 13566 rrna 6269992 . . . 6271519 + rrne 16s + y gtgaatac sc7c7 a10 6271446 751 13566 rrna 6269992 . . . 6271519 + rrne 16s − n ccttcgac sc7c7 g07 6272082 1387 13568 rrna 6271800 . . . 6274919 + rrne 23s − n gtatacgg sc7c7 b10 272989 2294 13568 rrna 6271800 . . . 6274919 + rrne 23s + y gtgcgtaat sc7c7 h10 273051 2356 13568 rrna 6271800 . . . 6274919 + rrne 23s + y gccgaagt sc7c7 c11 273249 2554 13568 rrna 6271800 . . . 6274919 + rrne 23s + y ggtaagtc sc7c7 g05 6273281 2586 13568 rrna 6271800 . . . 6274919 + rrne 23s − n cctgtcggc sc7c7 e09 6273329 2634 13568 rrna 6271800 . . . 6274919 + rrne 23s + y tcaaacat sc7c7 a01 6273776 3081 13568 rrna 6271800 . . . 6274919 + rrne 23s − n cgctggtc sc7c7 e12 6273823 3128 13568 rrna 6271800 . . . 6274919 + rrne 23s − n ccttacgg sc7c7 g03 6274456 3761 13568 rrna 6271800 . . . 6274919 + rrne 23s − n ccttttatc sc7c7 e01 6274937 4242 − ? cagtggacg sc7c7 a04 6274964 4269 − ? ggttgttc sc7c7 e04 6275179 4484 + ? ttccgtcac sc7c7 e05 6275630 4935 13571 sco5746 , sc7c7 . 01 − n actgctgat 6275223 . . . 6275900 + hypothetical protein sc7c7 . 01 sc7c7 e02 6275925 5230 + ? ggcttgttc sc7c7 d11 6277043 6348 13573 sco5747 , sc7c7 . 02c − y ggcccgacc 6276106 . . . 6278856 − putative regulatory protein sc7c7 c05 6277305 6610 13573 sco5747 , sc7c7 . 02c + n ggtcgggac 6276106 . . . 6278856 − putative regulatory protein sc7c7 e08 6277864 7169 13573 sco5747 , sc7c7 . 02c + n ccgatgaac 6276106 . . . 6278856 − putative regulatory protein sc7c7 a05 6277949 7254 13573 sco5747 , sc7c7 . 02c − y gtgctgcag 6276106 . . . 6278856 − putative regulatory protein sc7c7 f03 6277949 7254 13573 sco5747 , sc7c7 . 02c + n gcgtagacc 6276106 . . . 6278856 − putative regulatory protein sc7c7 g06 6278026 13573 sco5747 , sc7c7 . 02c + n gcgtagacc 6276106 . . . 6278856 − putative regulatory protein sc7c7 a11 6278408 7713 13573 sc05747 , sc7c7 . 02c + n gccgaccgc 6276106 . . . 6278856 − putative regulatory protein sc7c7 h03 6278566 13573 sco5747 , sc7c7 . 02c + n gcgtggacc 6276106 . . . 6278856 − putative regulatory protein sc7c7 d06 6278734 8039 13573 sco5747 , sc7c7 . 02c − y gttctgtga 6276106 . . . 6278856 − putative regulatory protein sc7c7 d10 6279071 8376 + ? gatgaaggt sc7c7 b04 6281301 10606 13576 sco5748 , sc7c7 . 03 − n gccacacac 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 c04 6281495 10800 13576 sco5748 , sc7c7 . 03 + y ggtcacgcg 6279265 . . . 6284754 − putative sensory histidine kinase sc7c7 f02 6281668 10973 13576 sco5748 , sc7c7 . 03 − n cccttggcg 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 h02 6281898 11203 13576 sco5748 , sc7c7 . 03 + y gtccaggtg 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 a09 6282182 11487 13576 sco5748 , sc7c7 . 03 − n cacctgacc 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 g02 6282297 11602 13576 sco5748 , sc7c7 . 03 + y gaccagctc 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 a07 6283214 12519 13576 sco5748 , sc7c7 . 03 + y ccagtcgtc 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 h11 6283268 12573 13576 sco5748 , sc7c7 . 03 − n gttctgctg 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 b06 6283391 12696 13576 sco5748 , sc7ct . 03 − n gttctgctg 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 c01 6284449 13754 13576 sco5748 , sc7c7 . 03 + y agcacggac 6279265 . . . 6284754 + putative sensory histidine kinase sc7c7 d07 6284914 14219 + ? acgtacggg sc7c7 h09 6286360 15665 13583 sco5750 , sc7c7 . 05 + y gtcttccgc 6286097 . . . 6288886 + ftsk homolog sc7c7 b03 6286832 16137 13583 sco5750 , sc7c7 . 05 + y cggccaccc 6286097 . . . 6288886 + ftsk homolog sc7c7 f05 6287186 16491 13583 sco5750 , sc7c7 . 05 + y tcgccgacc 6286097 . . . 6288886 + ftsk homolog sc7c7 d03 6287254 16559 13583 sco5750 , sc7c7 . 05 − n gcaccggcg 6286097 . . . 6288886 + ftsk homolog sc7c7 a08 6288131 17436 13583 sco5750 , sc7c7 . 05 + y acttcaacc 6286097 . . . 6288886 + ftsk homolog sc7c7 f11 6288234 17539 13583 sco5750 , sc7c7 . 05 − n ggccagctc 6286097 . . . 6288886 + ftsk homolog sc7c7 f09 6288477 17782 13583 sco5750 , sc7c7 . 05 + y cttcctgcc 6286097 . . . 6288886 + ftsk homolog sc7c7 b02 6289171 18476 + ? cgctcgaaa sc7c7 d08 6289754 19059 13587 sco5751 , sc7c7 . 06 − n ggcttgggg 6289190 . . . 6290053 + putative membrane protein sc7c7 h04 6290058 19363 + ? gcggggacc sc7c7 c03 6290188 19493 13588 sco5752 , sc7c7 . 07 − n ggcgcagcc 6290145 . . . 6291626 + conserved hypothetical protein sc7c7 . 07 sc7c7 f07 6291027 20332 13588 sco5752 , sc7c7 . 07 + y acttcgacc 6290145 . . . 6291626 + conserved hypothetical protein sc7c7 . 07 sc7c7 e11 6291102 20407 13588 sco5752 , sc7c7 . 07 + y tcctggagc 6290145 . . . 6291626 + conserved hypothetical protein sc7c7 . 07 sc7c7 e03 6291533 20838 13588 sco5752 , sc7c7 . 07 − n ccatacgac 6290145 . . . 6291626 + conserved hypothetical protein sc7c7 . 07 sc7c7 b01 6292335 21640 13591 sco5753 , pgsa 6291623 . . . 6292414 + − n gtccaggcc phosphatidylglycerophosphate synthase sc7c7 b11 6293516 22821 + ? gttttcgca sc7c7 a06 6293979 23284 13598 sco5756 , sc7c7 . 11 − n gtccacgac 6293651 . . . 6294121 + hypothetical protein sc7c7 . 11 sc7c7 e10 6294345 23650 13601 sco5757 , sc7c7 . 12 − n gcgcagggc 6294128 . . . 6294394 + hypothetical protein sc7c7 . 12 sc7c7 c07 6294633 23938 13602 sco5758 , sc7c7 . 13 + y tggtcaagg 6294450 . . . 6294824 + putative transcriptional regulator sc7c7 d09 6295168 24473 13604 sco5759 , sc7c7 . 14 − n cggtgagcg 6295168 . . . 6295306 + hypothetical protein sc7c7 . 14 sc7c7 c09 6295384 24689 13605 sco5760 , sc7c7 . 15c + n gtgcgggcc 6295344 . . . 6296174 − dna glycosylase sc7c7 c08 6295844 25149 13605 sco5760 , sc7c7 . 15c + n gtccagcac 6295344 . . . 6296174 − dna glycosylase sc7c7 b05 6295934 25239 13605 sco5760 , sc7c7 . 15c − y gcgctggag 6295344 . . . 6296174 − dna glycosylase sc7c7 d02 6295943 25248 13605 sco5760 , sc7c7 . 15c + n cgcgaacac 6295344 . . . 6296174 − dna glycosylase sc7c7 h06 6296183 25488 − ? cccttgagt sc7c7 g04 6296468 25773 13606 sco5761 , sc7c7 . 16c + n ggagcccgc 6296193 . . . 6301265 − putative atp − dependent dna helicase sc7c7 b08 6296627 25932 13606 sco5761 , sc7c7 . 16c − y gtacgacac 6296193 . . . 6301265 − putative atp − dependent dna helicase sc7c7 a03 6297137 26442 13606 sco5761 , sc7c7 . 16c − y cgaggagag 6296193 . . . 6301265 − putative atp − dependentdna helicase sc7c7 c02 6298130 27435 13606 sco5761 , sc7c7 . 16c + n cgtgaaggg 6296193 . . . 6301265 − putative atp − dependent dna helicase sc7c7 c06 6300217 29522 13606 sco5761 , sc7c7 . 16c − n gagcaggc 6296193 . . . 6301265 − putative atp − dependent dna helicase sc7c7 f01 6301309 30614 − ? gccacgccc sc7c7 g09 6302647 417 13616 sco5763 , sc4h8 . 02 − n cgggagggc 6302319 . . . 6303089 + putative membrane protein sch4h8 . 02 sc7c7 b07 6303689 1459 13619 sco5765 , sc4h8 . 04c + n agcacggcg 6303403 . . . 6304429 − hypothetical protein sch4h8 . 04c sc7c7 g01 6304465 2235 13621 sco5766 , sc4h8 . 05 − n ccgtcaacc 6304454 . . . 630468 + hypothetical protein sch4h8 . 05 sc7c7 g12 6304465 2235 13621 sco5766 , sc4h8 . 05 − n ccgtcaacc 6304454 . . . 630468 + hypothetical protein sch4h8 . 05 ausubel f m , brent r , kingston r e , moore d d , seidman j g , smith j a , struhl k ( 1989 ) current protocols in molecular biology . greene publishing associates and wiley - interscience , john wiley & amp ; sons , ny . bennett p m , grinsted j , richmond m h ( 1977 ) mol . gen . genet ., 154 , 205 - 211 . bentley , s . d ., et al . 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