• Novel missense germ line DDX41 mutations define an earlier age of onset of hematologic malignancies than loss-of-function alleles.• Carriers of DDX41 germ line mutations usually have normal blood counts until a myeloid or lymphoid malignancy develops.Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

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A gene coding for a thermostable lipase of Pseudomonas fluorescens SIK W1 was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. Twenty colonies with esterase activity on the tributyrin agar plate were isolated by screening the constructed Pseudomonas fluorescens genomic library. Only one out of the esterase positive 20 colonies had lipase activity on the agar plate containing olive oil and Rhodamine-B. The complete nucleotide sequence of the lipase gene was identified. The lipase gene consists of an open reading frame, 1347bp long, commencing with an ATG start codon encoding a polypeptide of 449 amino acid residues and a TGA stop codon. Comparison of this lipase amino acid sequence with those from another organisms sequenced to data showed the presence of the short homologous region Gly-X-Ser-X-Gly.

A thermostable lipase gene from Pseudomonas fluorescens S I K Wl was overexpressed in Escherichia coli BL21 using expression vector pTTY2. The amount of lipase produced by E. coli BL21 with pTTY2 was more than 40% of the total cell proteins when induced with isopropyl-fl-D-thiogalactopyranoside. The lipase was produced as inclusion bodies in the cytoplasm of E. coli. They were solubilized by 8 M urea and refolded into biologically active form. The refolded lipase showed high thermostability; the time required for 90% inactivation of the enzyme (D-value) was 4 h at 95 ° C and the increment of temperature to reduce heating times by 90% (zD value) was 76 ° C.

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