Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120.

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.

The sigE gene is required for normal expression of heterocyst-specific genes in Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 produces specialized cells for nitrogen fixation called heterocysts. Previous work showed that the group 2 sigma factor sigE (alr4249; previously called sigF) is upregulated in differentiating heterocysts 16 h after nitrogen step-down. We now show that the sigE gene is required for normal heterocyst development and normal expression levels of several heterocyst-specific genes. Mobility shift assays showed that the transcription factor NtcA binds to sites in the upstream region of sigE and that this binding is enhanced by 2-oxoglutarate (2-OG). Deletions of the region containing the NtcA binding sites in P(sigE)-gfp reporter plasmids showed that the sites contribute to normal developmental regulation but are not essential for upregulation in heterocysts. Northern RNA blot analysis of nifH mRNA revealed delayed and reduced transcript levels during heterocyst differentiation in a sigE mutant background. Quantitative reverse transcription-PCR (qRT-PCR) analyses of the sigE mutant showed lower levels of transcripts for nifH, fdxH, and hglE2 but normal levels for hupL. We developed a P(nifHD)-gfp reporter construct that showed strong heterocyst-specific expression. Time-lapse microscopy of the P(nifHD)-gfp reporter in a sigE mutant background showed delayed development and undetectable green fluorescent protein (GFP) fluorescence. Overexpression of sigE caused accelerated heterocyst development, an increased heterocyst frequency, and premature expression of GFP fluorescence from the P(nifHD)-gfp reporter.

Study on association of human leukocyte antigen-DRB1 alleles amongst Bangladeshi patients with rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic, systemic and autoimmune disease affecting 0.5-1% of the world population. Genetic and environmental factors are already established as being involved in the development of RA. Different human leukocyte antigen (HLA)-DRB1 alleles have major pathogenic effects to the development of RA. OBJECTIVE: To determine the HLA-DRB1 allelic frequency of RA in one Bangladeshi tertiary care center. METHODS: This case-control study was conducted at the Microbiology and Rheumatology Department of Bangabandhu Sheikh Mujib Medical University (BSMMU). Fifty-two patients diagnosed as having RA and 52 healthy controls were enrolled. Buccal swabs were collected from all subjects and HLA-DRB1 typing was carried out with polymerase chain reaction with sequence-specific primers (PCR-SSP) of low resolution. Blood was also collected for auto-antibodies (rheumatoid factor [RF] and anti-cyclic citrullinated peptide [anti-CCP]) detection from all subjects. RF was detected by nephelometry and anti-CCP was detected by using the enzyme-linked immunosorbent assay method. Statistical associations of HLA antigen between the groups were determined by chi-square test. RESULTS: In RA patients DR*04 and DR*10 were found at the DRB1 locus at higher frequencies (20.5%, P = 0.0035 and 18.3%, P = 0.0045, respectively). However, the frequency of DR*15 was significantly lower (P = 0.005) in RA cases (18.3%) than the control group (35.6%). The frequencies of autoantibodies (anti-CCP and RF) were compared between approximate shared epitope (SE) positive and SE negative patients, and no significant association was found. CONCLUSIONS: In this study DRB1*04 and DRB1*10 alleles were significantly associated with RA patients while DRB1*15 was found more in the control group.