A simple method for chloroplast transformation in Chlamydomonas reinhardtii.

Photosystem I (PSI) is a multisubunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photosynthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of Photosystem I. C. reinhardtii is also a particularly favorable organism for chloroplast transformation because it contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adenyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This article describes the procedures for chloroplast transformation.

Clinico-biological features of 30 patients with acute promyelocytic leukemia and response to combination induction chemotherapy with all-trans retinoic acid and anthracycline.

30 adult patients with acute promyelocytic leukemia (APL) were seen at our institution overthe past 7 years. Their white cell count at presentation ranged from 400/microl to 54,900/microl. Cytogenetic studies were successful in 28 patients, of which 26(93%) were positive for t(15;17). Molecular analysis by reverse-transcription polymerase chain reaction demonstrated the PML-RARalpha fusion transcript in all 30 patients. The majority of patients had breakpoints at the 3' end with bcr1 products predominating. Complete remission rate of 92% was achieved using all-trans retinoic acid and anthracycline as induction chemotherapy in 26 patients. Of these, retinoic acid syndrome was observed in 4 cases, with 1 fatality. In conclusion, APL is a distinct entity with a highly specific molecular marker - t(15;17) translocation - that can be successfully induced into remission with all-trans retinoic acid and anthracycline in most patients.

Philadelphia positive acute lymphoblastic leukaemia (ALL): clinico-haematologic characteristics, molecular analyses and 3-year follow up--a single institution study.

INTRODUCTION: The Philadelphia chromosome is one of the commonest chromosomal aberration in adult acute lymphoblastic leukaemia (ALL) patients. We present the results of a 3-year prospective study to look at the clinico-haematologic, immunophenotypic, cytogenetic and molecular profile of 13 adult patients with Philadelphia (Ph) positive ALL out of 35 newly diagnosed ALL seen at our institution over the past 3 years. MATERIALS AND METHODS: Thirty-five adult ALL patients seen between 1996 and 1998 comprised the study group. Marrow samples were obtained for immunophenotyping and karyotypic analysis at diagnosis. Samples were also obtained simultaneously for molecular testing for Ph chromosome. RESULTS: Thirteen patients were found to be Ph positive by molecular analysis while cytogenetic studies identified the chromosomal abnormality in 9 of these patients. The median age of our Ph positive patients was similar to those without Ph chromosome. Pre-B phenotype appears to be common in this group of patients. In concordance with other studies, Ph positive ALL was associated with a poor prognosis in our patients. CONCLUSION: Identification of Ph chromosome is important in the management of patients with ALL as it is an important prognostic marker.

Molecular and phenotypic spectrum of de novo Philadelphia positive acute leukemia.

The Philadelphia chromosome is present in a heterogeneous group of leukemias. It is most commonly associated with chronic myelogenous leukemia (CML) and B-lineage acute lymphoblastic leukemia (ALL) being found in more than 95% and 15-25% of cases respectively. We undertook a study to determine the morphologic, phenotypic and molecular diversity of Philadelphia positive de novo acute leukemia patients seen at our institution over the past 3 1/2 years. Twenty-one patients with de novo acute leukemia were found to have the Philadelphia chromosome by cytogenetic studies. They consisted of 3 patients with acute myelogenous leukemia (AML), 1 biphenotypic leukemia and 17 ALL patients. Of the patients with ALL, 16 were of B-lineage while 1 had a T-cell phenotype. Ten patients expressed the p210 BCR-ABL transcript alone and 10 expressed only the p190 BCR-ABL transcript. One patient had co-expression of p190 and p210 b3a2 BCR-ABL transcripts. Thus the Philadelphia chromosome can be found in a diverse cohort of morphologic and immunologic subtypes of de novo acute leukemia reflecting the heterogeneity of lineage involvement in this disease.