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Biotechnology Homework Help: Chloroplast DNA Integration

Chloroplast transformation or transgene integration into cpDNA (chloroplast DNA) has been quite successful in the green alga Chlamydomonas. Two transformation methods have been successfully employed: (i) particle gun and (ii) agitation of cells (devoid of cell wall) in the presence of glass beads and DNA. Selection of transformants utilizes specific selectable markers located in cpDNA, e.g. spectinomycin and streptomycin resistance etc. According to i need help with homework experts there are a couple of reports of gene integration in cpDNA in higher plants. In one study using Agrobacterium-mediated gene transfer into the nuclear genome, a single tobacco plant showed integration of the transgene (CAT) into cpDNA. This gene integration was accidental and not by design. Subsequently a plastid transformation vector pZS148 was devised, pZS148 contains the following modules: (i) 16 S ribosomal DNA (rDNA) from the tobacco genotype SPC2; (it specifies resistance to both streptomycin and spectinomycin), plasmid vector (pBluescript IKS +) for cloning in E. coli. This vector has been designed as a shuttle vector to replicate both in E. coli and chloroplasts. The vector pZS148 was introduced into tobacco leaves by particle gun delivery, and 3 transformants were recovered. These plants showed material inheritance for reistance to the two antibiotics. It was estimated that gene integration in cpDNA was only 1% of that in nuclear DNA. This may be due to the large number (ca 3000-12000) of copies of the plastid genome in each cell, the lack of a suitable selectable marker which will allow survival due to and multiplication of a single transformed chloroplast genome, and the nonavailability of a guidance system for the DNA into the chloroplasts. It has been suggested that more efficient DNA delivery systems may be developed for chloroplasts using the following approaches: (i) naked DNA may be introduced into plant protoplasts by microinjection, electroporation or PEG-mediated delivery; the chloroplasts being similar to bacteria may be expected to take up this DNA. This may be greatly facilitated of this DNA contains a sequence which is recognized by a receptor on the chloroplast surface. (ii) DNA could be microinjected into the chloroplasts in vivo. Alternatively, according to studydaddy (iii) chloroplasts may be isolated, induced to take up DNA and returned into the cells. The above proposed methods are yet to be demonstrated, and a directed cpDNA/mDNA modification, therefore, must await future developments.

Biotechnology Homework Help: Chromosome Jumping and Walking

Physical map of chromosome is constructed by digesting the genomic DNA with Not I and separating the fragments by pulse field gradient electrophoresis. Each clone of the jumping and the linking libraries is hybridized with all the DNA fragments so obtained. Each clone should hybridize with two fragments which contain the sequences represented in the clone. According to the data from the jumping library and linking library are pooled together to determine the correct order of the various fragments in the organisation of different chromosomes in the genome.

Chromosome walking

This technique is a more conventional approach and is used to prepare restriction maps of small regions of chromosomes. It also helps in identifying DNA fragments having overlapping regions, and as a result to locate two or more fragments that may contain a large sequence of interest, e.g. parts of a single large gene (some eukaryotic genes may be over 100 kb long). The technique uses conventional cloning techniques to obtain DNA fragments. First, a single DNA fragment is identified to serve as the reference point. The restriction map of this fragment is prepared and one end of it is used as a probe to locate a neighbouring overlapping fragment. Restriction map of this new fragment is prepared and its other end is used as a probe to locate the next overlapping neighbour fragment.

The techniques of chromosome jumping and chromosome walking enable the identification of contigs. Contigs (contiguous fragments) are clones containing neighbouring DNA fragments having an overlapping region. Contigs greatly facilitate mapping and, ultimately, the correct alignment of base sequence data.

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