Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae.
Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of slashed circleC31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNA(Gln) amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also.
Santhosh
Ramachandran Sarojini
RS
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Palkalainagar, Madurai 625 021, India.
Pandian
Shunmugiah Karutha
SK
Lini
Nirmala
N
Shabaana
Abdul Khader
AK
Nagavardhini
Avuthu
A
Dharmalingam
Kuppamuthu
K
D001287:Attachment Sites, Microbiological / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006801:Humans; D015440:Leprosy, Lepromatous / Q000382:microbiology; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000235:genetics* / Q000472:pathogenicity; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009876:Operon; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; C473802:mammalian cell entry protein Mce1A, Mycobacterium tuberculosis