Development of a plasmid-mediated reporter system for in vivo monitoring of gene expression in the archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.     

Fanconi anemia: genes and function(s) revisited

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.     

Distinct subsets of primary effusion lymphoma can be identified based on their cellular gene expression profile and viral association

Primary effusion lymphomas (PELs) are specifically associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection and most frequently occur in human immunodeficiency virus (HIV)-positive individuals as lymphomatous effusions in the serous cavities without a detectable solid tumor mass. Most PELs have concomitant Epstein-Barr virus (EBV) infection, suggesting that EBV is an important pathogenetic cofactor, although other as yet unidentified cofactors, such as cellular genetic alterations, are also likely to play a role. Lymphomatous effusions that lack KSHV also occur; these are frequently EBV associated in the setting of HIV infection. Here we used gene expression profile analysis to determine the viral impact on cellular gene expression and the pathogenesis of these lymphomatous effusions. Our results show that many genes, including cell cycle and signal transduction regulators, are differentially expressed between KSHV-positive PELs and KSHV-negative lymphomatous effusions and also between KSHV-positive, EBV-positive and KSHV-positive, EBV-negative PELs. Our results confirm that KSHV plays an important role in the pathogenesis of PELs, as its presence selects for a very distinct cellular gene expression category and a clearly different lymphoma type. Within the KSHV-positive PELs, the effect of EBV is more subtle but nevertheless clear.     

Extrapulmonary Tuberculosis: Mycobacterium tuberculosis Strains and Host Risk Factors in a Large Urban Setting in Brazil

Background

Factors related to the development of extrapulmonary forms of tuberculosis (EPTB) are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB) strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil.

Methods and Findings

We conducted a retrospective laboratory-based surveillance study of new tuberculosis (TB) cases diagnosed in Espírito Santo state, Brazil between 1998 and 2007. We genotyped 612 isolates of MTB from 606 TB patients using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing and compared sociodemographic and clinical characteristics of patients with pulmonary TB (PTB) and EPTB. Among 606 patients, 464 (77%) had PTB, 79 (13%) had EPTB, 51 (8%) had both, and 12 (2%) had miliary TB. The IS6110 RFLP analysis demonstrated that 250 (41%) isolates belonged to clustered RFLP patterns, 27 (11%) of which were from EPTB. We identified 73 clusters including 35 (48%) composed of 2 isolates each. By spoligotyping, 506 (83%) MTB isolates fell into known patterns and 106 (17%) fell into patterns with no family assignment; 297 (48%) isolates belonged to the Latin-American Mediterranean family. Higher school level (4-7 years OR: 0.16 95% CI 0.34-0.73 and > 8 years of education, OR 0.06 95% CI 0.009-0.50) white ethnicity (OR: 2.54 95% CI 1.03-6.25) and HIV infection (OR: 16.83 95% CI 5.23-54.18) were associated with EPTB. No specific strain lineage or percentage of clustering was associated with EPTB.

Conclusions

These results demonstrate that risk factors for EPTB are related more to host than to MTB strain lineage characteristics.     

BOOK REVIEWS

Book reviewed in this article:
Dravidian Borrowings from Indo-Aryan. M. B. Emeneau and T. Burrow.
Brahui and Comparative Dravidian Grammar. M. B. Emeneau.