Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome.

Abstract

As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.

Synthetic Botany

“…As a consequence, the risk of inconsistent gene expression by the positional effect in T-DNA-mediated nuclear transformation is alleviated in the chloroplast environment (Daniell et al 2002). The high efficiency of homologous recombination in the chloroplast (Blowers et al 1989) also allows the simultaneous introduction of modifications to several sites of the chloroplast genome by means of cotransformation (Kindle et al 1991), encouraging the implementation of phenotypic traits based on multiple foreign genes. Multiple genes may be conveniently organized in operon-like polycistronic units (Hasunuma et al 2008), which can be processed into more efficiently translated monocistronic transcripts by the incorporation of intercistronic expression elements (Lu et al 2013).…”

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

“…The Dral and Seal sites between the antibiotic resistance genes are such potential insertion sites in plasmid pJS75. Insertion of foreign genes into the Chlamydomonas plastid genome has already been reported by Blowers et al (1989) and Goldschmidt-Clermont (1991). In the primary calli, almost all of the pJS75 markers were recovered in homoplasmic form.…”

“…These objectives have recently been achieved in the unicellular alga Chlamydomonas (Boynton et al, 1988;Blowers et al, 1989Blowers et al, , 1990Goldschmidt-Clermont, 1991;Goldschmidt-Clermont et al, 1991;Kindle et ai., 1991, Newman et al, 1991Przibilla et al, 1991;Roffey et al, 1991;. Replacement by transformation of the gene of the 16s rRNA in tobacco, a land plant, has also been reported by Svab et al (1990).…”

Evaluation of Spirulina platensis Resistance to Different Antibiotics to Find a Selectable Marker for Genetic Transformation

“…These gene can be expressed by the use of suitable prokaryotic expression elements including promoter, terminator etc. The mentioned elements should be preferably isolated and set on the upstream and downstream of the inserted gene expressed (26 …”