Plasmids

From Scott Newman, Boynton-Gillham laboratory, October 1990

chloroplast DNA XhoI-SmaI (2.8 kb) in BSKS+, 5′ exon of the 23S rRNA gene

host strain: JM109
amp resistant

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Strain: partly derived from CC-125 and partly from the Rochaix cw15 strain

Insert: XhoI-SmaI (2.6 kb)

Genes present: 23S rRNA, 5′ exon

Vector: BSKS+

Bacterial host strain: JM109

Selectable marker: amp-r, white on X-gal

Origin: Scott Newman in Boynton/Gillham lab, 1990

The insert was constructed by ligating the 1.1 kb XhoI-BamHI fragment from Bam 14/15 (P-14) to the 1.5 kb BamHI-SmaI fragment from Bam 11/12 (P-59), resulting in a 2.6 kb Xho-Sma fragment.

From Steve Mayfield to Boynton-Gillham laboratory, 1990

Chloroplast DNA EcoRI-BamHI (4.5 kb) in BSSK-; psbA cDNA (no introns)

host strain: JM109
amp resistant

Johanningmeier U, Heiss S (1993) Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene. Plant Mol Biol 22:91-99

Boynton-Gillham laboratory, Duke University, 1990

Chloroplast DNA XhoI 8 (9.5 kb) in BSKS+; psbA through 3′ exon of 23S; ca. 1.5 kb Kpn fragment was deleted between psbA and 5S;; contains er-u-1a erythromycin resistance mutation in 23S, from CC-64

host strain: JM109
amp resistant

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Strain: CC-64 er-u-1a mt+

Insert: XhoI 8 ΔKpnI (9.5 kb)

Genes present: psbA, 5S rRNA, 23S rRNA (3′ exon)

Vector: BSKS+

Bacterial host strain: JM109

Selectable marker: amp-r, white on X-gal

Origin: Amnon Lers in Boynton/Gillham lab, 1990

The insert is a subclone of Xho 8 (P-259B) in which three KpnI fragments including the BamHI site (approx. 1.5 kb) located between the psbA and 5S genes were deleted and the remaining two Kpn ends were religated, leaving only one Kpn site.

The er-u-1a mutation is a C->T change at bp 2622 in C. reinhardtii 23S rRNA gene (Lemieux numbering, equivalent to E. coli 2611), which confers erythromycin resistance in Chlamydomonas.

Boynton-Gillham laboratory, Duke University, 1990

Chloroplast DNA Kpn I (3.1 kb) in BSKS+; psbA exons 4 and 5 with R238K mutation in exon 4 and DCMU4 (S264K) in exon 5, from CC-2473

host strain: JM109
amp resistant

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Strain: CC-2473 DCMU4

Insert: KpnI (3.1 kb)

Genes present: psbA exons 4 and 5

Vector: BSKS+

Bacterial host strain: JM109

Selectable marker: amp-r, white on X-gal

Origin: Amnon Lers in Boynton/Gillham lab, 1990

This plasmid was derived from P-260 by in vitro mutagenesis of the arg residue at aa 238 to lys.

DCMU4 is a ser->ala mutation at aa 264 in the C. reinhardtii psbA 5th exon, conferring resistance to DCMU and atrazine.

Boynton-Gillham laboratory, Duke University, 1990

Chloroplast DNA Kpn I (3.1 kb) in BSKS+; psbA exons 4 and 5 with R238K mutation in exon 4

host strain: JM109
amp resistant

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Insert: KpnI (3.1 kb)

Genes present: psbA (exons 4 and 5)

Vector: BSKS+

Bacterial host strain: JM109

Selectable marker: amp-r, white on X-gal

Origin: Amnon Lers in Boynton/Gillham lab, 1990

A 0.9 kb Eco RI fragment from P-260A (Eco sites located within 4th intron of psbA and BSKS polylinker) containing psbA exon 4 was mutated in vitro at position 238 arg -> lys and cloned in the EcoRI site of P-299 (2.3 kb KpnI-EcoRI fragment containing wild type sequences of psbA intron 4 and exon 5 cloned into BSKS+), yielding a 3.1 kb Kpn fragment inserted into BSKS+. This plasmid has the psbA Kpn 3.1 kb fragment with only the arg -> lys mutation.

From René Matagne to Boynton-Gillham laboratory, 1991

Mitochondrial DNA EcoRI-HindIII (1.5 kb) in pUC12; contains nd4 and cob genes

host strain: unknown
amp resistant

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA KpnI (2.4 kb) in pUC18; 16S rRNA gene with spr-u-1-H-4 and nr-u-2-1 mutations

host strain: JM109
amp resistant

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Strain: CC-245 nr-u-2-1 spr-u-1-H-4

Insert: KpnI (2.4 kb)

Genes present: 16s rRNA

Vector: pUC18

Bacterial host strain: JM109

Selectable marker: amp-r, white on X-gal

Origin: Scott Newman in Boynton/Gillham lab, 1991

The nr-u-2-1 mutation is an A->G change at bp 1340 in the 16S rRNA gene, and the spr-u-1-H-4 is an A->G change at bp 1123 in the same gene, conferring resistance to neamine/kanamycin and spectinomycin, respectively. See Harris et al., Genetics 123: 281-292 (1989).

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA EcoRI-XhoI (5.7 kb) in BSKS+; psbA exon 5 with DCMU4 mutation, 5S, and 23S 3′ end with er-u-1a mutation

host strain: JM109
amp resistant

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Strain: CC-2534

Insert: chloroplast DNA EcoRI-XhoI (5.7 kb)

Genes present: psbA (intron 4, exon 5); 5S rRNA, 23S rRNA (3’ end)

Vector: BSKS+

Bacterial host strain: JM109

Selectable marker: amp-r, white on X-gal

Origin: Newman, 1/1991

CC-2534 = DCMU-4 er-u-1a. DCMU-4 mutation in 5th exon of C. reinhardtii psbA gene causes ser -> ala change at aa 264 D1 protein, conferring resistance to DCMU and atrazine; er-u-1a mutation is C ->T change at bp 2622 in C. reinhardtii 23S rRNA (Lemieux numbering, equivalent to E. coli 2611), conferring resistance to erythromycin.

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA BamHI-EcoRI (2.5 kb) in BSKS+, from a cw15 strain equivalent to CC-400; contains Eco 33 and part of Eco 28

host strain: JM83
amp resistant

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Strain: Rochaix cw15

Insert: BamHI-EcoRV (2.5 kb)

Vector: BSKS+

Bacterial host strain: JM83

Selectable marker: amp-r, white on X-gal

Origin: Charles Hauser in Boynton/Gillham lab, 1991

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA EcoRI-(XhoI) (3.6 kb) in pUC18; psbA exon 5 with DCMU4 mutation, 5S and 23S 3′ end; Kpn deletion between psbA and 5S

host strain: JM109
amp resistant

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Strain: CC-2534 DCMU-4 er-u-1a

Insert: EcoRI-(XhoI) (3.6 kb)

Genes present: psbA – 23S rRNA, Kpn deletion

Vector: pUC18

Bacterial host strain: JM109

Selectable marker: amp-r

Origin: Scott Newman in Boynton/Gillham lab, 1991

Insert = 2.2 kb Eco-Kpn fragment from P-322 containing 5th exon of psbA, ligated to 1.4 kb Kpn-Sal fragment from P-14 (Sal site located in BSKS polylinker) containing 5S rRNA gene and small exon of 23S rRNA gene. Resulting construct has Kpn fragments between psbA and 5S rRNA deleted as in P-320, but has wild type (erythromycin sensitive) 23S gene derived from P-14.

The DCMU-4 mutation in 5th exon of C. reinhardtii psbA gene causes a ser -> ala change at aa 264 of D1 protein, conferring resistance to DCMU and atrazine.

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA KpnI-BamHI (2.6 kb) in pUC18; 5S and 3′ end of 23S with er-u-1a mutation

host strain: JM109
amp resistant

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Strain: CC-64 er-u-1a

Insert: KpnI-BamHI (2.6 kb)

Genes present: 23S rRNA, 3′ end

Vector: pUC18

Bacterial host strain: JM109

Selectable marker: amp-r

Origin: Scott Newman in Boynton/Gillham lab, 1991

This is a subclone of P-321, which was a ligation of sequences from CC-2534 and CC-64. The fragment remaining in this subclone derives mainly from CC-64.

The er-u-1a mutation is a C > T change at bp 2622 in C. reinhardtii 23S rRNA (Lemieux numbering, equivalent to E. coli 2611), conferring resistance to erythromycin.

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA BamHI 11/12 in a pUC18 deletion plasmid; contains 16S and 5′ end of 23S, spr-u-1-27-3 mutation in 23S rRNA, from CC-105

host strain: JM83
amp resistant

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Strain: CC-105 spr-u-1-27-3 mt+

Insert: Bam 11/12 (7.0 kb)

Genes present: 16S and 23S rRNA

Vector: pUC18 with Nde, Eco and Pst sites deleted

Bacterial host strain: JM83

Selectable marker: amp-r

Origin: Charles Hauser in Boynton/Gillham lab, 1991

The mutation in spr-u-1-27-3 is a G -> A change at bp 1125 in the 16S chloroplast ribosomal RNA.

From Pete Lefebvre to Boynton-Gillham laboratory, 1991

NIT1 genomic; Sal I/Bgl II (14.5 kb) in pUC119

host strain: DH5
amp resistant

Fernández E, Schnell R, Ranum LP, Hussey SC, Silflow CD, Lefebvre PA (1989) Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 86:6449-6453

Boynton-Gillham laboratory, Duke University, 1991

Chloroplast DNA KpnI-PstI (0.9 kb) in KS+; psbA exon 5, mutated to stop codon at aa 345

host strain: JM109
amp resistant

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Insert: KpnI-PstI (0.9 kb)

Genes present: psbA exon 5

Vector: KS+

Bacterial host strain: JM109

Selectable marker: amp-r

Origin: Amnon Lers in Boynton/Gillham lab, 1991

The KS+ vector was digested with KpnI + PstI and a Kpn-Pst insert of 0.9 kb which includes exon 5 of psbA was inserted. This exon was mutated in vitro to change the aa codon ser-345 to a TAA stop codon followed by another change to create a new AflII restriction site. The oligo used in this mutagenesis was psbAstop. This construct was used to obtain the NP mutant.

Lers A, Heifetz PB, Boynton JE, Gillham NW, Osmond CB (1992) The carboxyl-terminal extension of the D1 protein of photosystem II is not required for optimal photosynthetic performance under CO2- and light-saturated growth conditions. J Biol Chem 267:17494-17497

From Jean-David Rochaix to Boynton-Gillham laboratory, 1991

ARG7 genomic, 7.1 kb BamHI-SalI in pBR329; can rescue arg2 and some other arg7 mutants

host strain: unknown
amp resistant

Debuchy R, Purton S, Rochaix JD (1989) The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J 8:2803-2809

From Michel Goldschmidt-Clermont to Boynton-Gillham laboratory, 1992

Chloroplast DNA expression vector; aadA flanked by Chlamy 5′ atpA and 3′ rbcL; EcoRI-SacI (1.9 kb) in pUC18

host strain: JM83
amp resistant

Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucl Acids Res 19:4083-4089; also see P-484

Boynton-Gillham laboratory, Duke University, 1992

Chloroplast DNA BamHI 10 (7.6 kb) in KS+; contains atpB gene; same insert as P-17 but in KS+ vector

host strain: XLI-Blue
amp resistant

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Strain: Rochaix cw15

Insert: Bam HI 10 (7.6 kb)

Genes present: atpB

Vector: KS+

Bacterial host strain: XLI-Blue

Selectable marker: amp-r

Origin: Charles Hauser in Boynton/Gillham lab, 1991

This is the Bam 10 fragment from P-17 cloned into KS+.

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

From Monique Turmel to Boynton-Gillham laboratory, 1992

Chloroplast DNA EcoRI 30 (1.5 kb) in KS-; contains ycf3 and most of ycf4

host strain: unknown
amp resistant

From Karen Kindle to Boynton-Gillham laboratory, 1992

CabII-1 genomic, KpnI-EcoRI (7.8 kb) in p1B176

host strain: JM83
amp resistant

Imbault P, Wittemer C, Johanningmeier U, Jacobs JD, Howell SH (1988) Structure of the Chlamydomonas reinhardtii cabII-1 gene encoding a chlorophyll-a/b-binding protein. Gene 73:397-407

From Karen Kindle to Boynton-Gillham laboratory, 1992

CabII-1 genomic, pSP-CAB; 2.4 kb EcoRI-HindIII fragment in pSP65

host strain: JM83
amp resistant

Imbault P, Wittemer C, Johanningmeier U, Jacobs JD, Howell SH (1988) Structure of the Chlamydomonas reinhardtii cabII-1 gene encoding a chlorophyll-a/b-binding protein. Gene 73:397-407 Nikaido SS, Locke CR, Weeks DP (1994) Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii. Plant Mol Biol 26:275-284

From Karen Kindle to Boynton-Gillham laboratory, 1992

3′ UTR of cabII-1, 0.3 kb in pSP65; digestion with HindIII allows synthesis of an antisense RNA

host strain: JM83
amp resistant

Imbault P, Wittemer C, Johanningmeier U, Jacobs JD, Howell SH (1988) Structure of the Chlamydomonas reinhardtii cabII-1 gene encoding a chlorophyll-a/b-binding protein. Gene 73:397-407

Boynton-Gillham laboratory, Duke University, 1992

Chloroplast DNA PstI 15 (3.1 kb), vector uncertain; contains rbcL (all but extreme 3′ end), atpA (5′ half)

host strain: unknown
amp resistant

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Strain: CC-125 wild type mt+

Insert: PstI 15 (3.1 kb)

Genes present: rbcL, atpA

Vector: unknown

Bacterial host strain: unknown

Selectable marker: amp-r

Origin: Boynton/Gillham lab, ca. 1992

The insert came from P-83, which was tetracycline resistant, and was cloned into an ampicillin resistant vector by a student in the Boynton/Gillham lab. The file notes don’t specify which vector or host strain, however.

Keith Kozminski and Joel Rosenbaum to Boynton-Gillham laboratory, 1992

Alpha-tubulin with RBCS2 constitutive promoter (1.1 kb)

host strain: XL-1 blue
amp resistant

Michel Goldschmidt-Clermont to Boynton-Gillham laboratory, 1992

Chloroplast aadA cassette, KpnI-SacI (4.8 kb) in BSKS-; tscA, 5′ rbcL : aadA : 3′ rbcL

host strain: JM83
amp resistant

Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucl Acids Res 19:4083-4089

Michel Goldschmidt-Clermont to Boynton-Gillham laboratory, 1992

Chloroplast aadA cassette, KpnI-SacI (5 kb) in BSKS-; tscA, 5′ atpA : aadA : 3′ rbcL

host strain: JM83
amp resistant

Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucl Acids Res 19:4083-4089

Boynton-Gillham laboratory, Duke University, 1992

Chloroplast DNA GUS cassette, in KS+; contains the entire atpB 5′ UTR and promoter

host strain: JM83
amp resistant

Boynton-Gillham laboratory, Duke University, 1992

Chloroplast DNA cassette containing recA from E. coli with internal deletion, flanked by atpA 5′, rbcL 3′; in BSKS+; ca. 2.04 kb

host strain: XL1-Blue
amp resistant

Boynton-Gillham laboratory, Duke University, 1992

Chloroplast DNA cassette containing recA from E. coli with N-end deletion, flanked by atpA 5′, rbcL 3′; in BSKS+; ca. 2.01 kb

host strain: XL1-Blue
amp resistant

Boynton-Gillham laboratory, Duke University, 1992

Chloroplast DNA expression vector: aadA flanked by atpA 5′, rbcL 3′; EcoRI-BamHI (1.9 kb); same insert as P-423, but in BSKS+

host strain: XL1-Blue
amp resistant

Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucl Acids Res 19:4083-4089

From Christoph Beck to Boynton-Gillham laboratory, ca. 1992

HSP70C genomic, = Beck pCB470; 4 kb SalI-BamHI fragment in BSKS+

host strain: JM83 r-
amp resistant

von Gromoff ED, Treier U, Beck CF (1989) Three light-inducible heat shock genes of Chlamydomonas reinhardtii. Mol Cell Biol 9:3911-3918