Unknown,Transcriptomics,Genomics,Proteomics

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Microarray-based transcription profiling of Streptococcus pneumoniae R6 delta rgtA ,delta rgtD and delta cpoA

ABSTRACT: Alterations in genes for penicillin-binding proteins (pbp) are well-known determinants for the resistance of Streptococcus pneumoniae to B-lactam antibiotics. Surprisingly, some mutations in non-pbp genes were also found to contribute to B-lactam resistance. Two of them discovered in the piperacillin resistant mutants P106 and P104, affect the expression of cpoA (encoding a glycosyltransferase) and of the rgtABCDHR cluster (encoding two small membrane proteins, an ABC transporter and a regulatory two-component system), respectively. cpoA and rgtABCDHR are involved in maintaining the synthesis and the proper ratio of the two major membrane glycolipids, and deletions in these genes led to complex phenotypes. In attempts to identify genetic determinants for these phenotypes, the global trancription patterns of the deletion mutants R6 delta cpoA, R6 delta rgtA and R6 delta rgtD were compared to that of the parent strain R6.

ORGANISM(S): Streptococcus pneumoniae

SUBMITTER: Martin Rieger

PROVIDER: E-MEXP-2923 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Head and neck squamous cell carcinomas (HNSCCs) is the seventh most common solid malignancy in the United States, accounting for more than 47,000 new cancer cases. Surgery of patients clinically diagnosed with lymph node metastasis (N+) also involves neck dissection, which causes disfigurement and pain. However, after histological examination, more than 30% of clinically N+ patients turn out to be metastasis-free (N0). Clinically negative lymph node patients have occult node metastasis in up to 50% of the cases. Within two years of follow-up these patients may or may not develop metastatic disease. On the other hand, around 50% of N0 patients do not have occult node metastasis. Therefore, many N0 patients undergo neck dissection unnecessarily. Due to limitations in detecting lymph node metastasis before surgery, both N+ and N0 patients may receive inappropriate treatment. This indicates that a better understanding of the biology of HNSCC is urgently needed. Despite tumor complexity, many studies have tried unsuccessfully to test single genes to be used as prognostic markers in HNSCC. A group of tumor samples can be characterized in terms of the behavior of modules. These modules include clusters of coexpressed genes, such as genes that belong to the same pathway or functional category. Cancer is a multifaceted phenomenon that involves activation and/or disruption of various cellular processes. Thus, the identification of pathways or group of genes such as those involved in the development of lymph node metastasis and recurrent disease may help understanding the complex biology of cancer. Samples were obtained during surgery and neck dissection of 81 patients with primary untreated HNSCC at the Head and Neck Surgery Department from the Hospital AC Camargo (São Paulo, Brazil) between 1998 and 2003. Diagnosis of HNSCC was determined by biopsy. All patients signed a pre-informed consent and the study was approved by our institutional review board. After surgery, all patients were treated with adjuvant radiotherapy. Tumor samples were snap-frozen in liquid nitrogen. Before RNA extraction, diagnosis was confirmed by hematoxylin-eosin staining. Frozen samples were hand dissected for removal of normal cells, necrosis, and infiltrating inflammatory cells. Total RNA was extracted using TRIzol. RNA quality was accessed by spectrophotometry and gel electrophoresis. To be considered as high-quality, the RNA had to have a 260/280 ratio higher than 1.7 and a 18S/28S rRNA ratio ~2. Amplifictaion of the mRNA was done using a T7-based protocol and cDNA was indirectly labeled with Alexa Dye 555 or 647. Samples and a common RNA reference were hybridized overnight at 42oC in dye-swap to a 4,800-element in-house printed microarray, enriched with cancer-related ESTs derived from the Human Cancer Genome Project. After washing, slides were scanned on a confocal laser scanner and data were extracted.

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Microarray-based transcription profiling of Streptococcus pneumoniae R6 delta rgtA ,delta rgtD and delta cpoA

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