The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

Production of correctly processed human serum albumin in transgenic plants.

We have used a modified CaMV 35S promoter to direct the expression of chimaeric genes encoding human serum albumin (HSA) in transgenic potato and tobacco plants. To secrete the protein, either the human prepro-sequence or the signal sequence from the extracellular tobacco protein PR-S was used. We demonstrate secretion of HSA with both types of signal sequences in transgenic leaf tissue and in suspension cultures. HSA produced in transgenic potato plants was purified to chromatographic homogeneity. N-terminal amino acid sequence analysis revealed that the processing of the precursor protein was dependent on the type of signal sequence. Expression of the human preproHSA gene lead to partial processing of the precursor and secretion of proHSA. Fusion of HSA to the plant PR-S presequence resulted in cleavage of the presequence at its natural site and secretion of correctly processed HSA that is indistinguishable from the authentic human protein.

Regulation of porcine heart and skeletal muscle AMP-deaminase by adenylate energy charge.

1. Cytosol from pig skeletal muscle, but not heart, contains an inhibitor of AMP-deaminase (AMP-D, EC 3.5.4.6) which reduces AMP-D activity 8-fold. 2. Heart and skeletal muscle AMP-D have been purified to apparent homogeneity by cellulose phosphate and DEAE-Sephacel chromatography. 3. AMP-D from skeletal muscle is inhibited more severely than the heart enzyme by an increase in adenylate energy charge to levels exceeding 0.4. Nevertheless both enzymes seem to be regulated by the energy charge, which contrasts with reports for rabbit heart AMP-D.

Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves.

Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.

Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.

As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all. Plant trehalase activity was shown to affect the accumulation of trehalose in these plants. An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants. Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N. tabacum development. It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.