Question

Describe how you can determine if the gene is interrupted and, if so, the number of interruptions by restriction endonuclease analysis and Southern analysis.

Answer 1

Interrupted gene contains expressed regions called exons, split with unexpressed regions called introns. Exons provide instructions for coding proteins, that create mRNA for the synthesis of proteins. Introns are removed by recognition of the donor site and splice acceptor site.It is determined by Restriction endonuclease and southern analysis.

The restriction endonucleases recognize specific base sequences in double-helical DNA and cleaves it at specific places, both strands containing the recognized sequences.They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating the genes, and creating new DNA molecules that can be cloned.They are found in a wide variety of prokaryotes. Their biological role is to cleave foreign DNA molecules. The cell's own DNA is not destroyed, because the sites recognized by its own restriction enzymes are methylated. Many restriction enzymes recognize specific sequences of four to eight base pairs and hydrolyze a phosphodiester bond in each strand .A striking characteristic of these cleavage sites is that they nearly possess twofold rotational symmetry. The recognized sequence is palindromic, or an inverted repeat, and the cleavage sites are symmetrically positioned.

Any small difference between related DNA molecules can be readily detected because their restriction fragments can be separated and displayed by gel electrophoresis. In many types of gels, the electrophoretic mobility of a DNA fragment is inversely proportional to logarithm of the number of base pairs, up to a limit. Polyacrylamide gels are used to separate fragments containing nearly 1000 base pairs, whereas more porous agarose gels are used to resolve mixtures of larger fragments (about 20 kb). An important feature of these gels is their high resolving power.Entire chromosomes containing millions of nucleotides can be separated on agarose gels by applying pulsed electric fields in different directions. Bands or spots of radioactive DNA in gels can be visualized by autoradiography . Alternatively, a gel can be stained with ethidium bromide, which fluoresces an intense orange when bound to double-helical DNA molecule.

Southern blot is then used by hybridizingwith a labeled probe specific to the cDNA (only exons).  The pattern of labeled fragments on the autoradiogram shows the fragments that contain exons.  Alignment of these with the restriction map gives an approximate position of the exons. The blot-hybridization approach can be combined with a PCR (polymerase chain reaction) analysis for higher resolution. Primers are synthesized that anneal to adjacent exons.  The product can be cloned and sequenced for more detailed information(to define the exon/intron junctions).

Subsequently, the nucleotide sequence of exonic regions and the entire gene is determined. The presence of introns were confirmed and their locations defines precisely in DNA sequences of isolated clones of the genes.

The southern blot analysis confirms the interruption of mpgS gene, using DNA from strains HB27 and RQ-1 and from the corresponding kanamycin-resistant mutants. Scheme of the genetic organization of mannosylglycerate-synthesizing genes in the chromosome of the wild-type T. thermophilus (wt) strains and in mutants after insertion of a kanamycin resistance cassette, with representation of primers used for PCR amplifications. Bands were located by hybridization with an 1,176 bp probe corresponding to the mpgS gene and with a 960 bp probe corresponding to the kat gene.