Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase.

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

Ca2+-calmodulin binds to the carboxyl-terminal domain of dystrophin.

The unique COOH-terminal domain of dystrophin (mouse dystrophin protein sequences 3266-3678) was expressed as a chimeric fusion protein (with the maltose-binding protein), and its binding to calmodulin was assessed. This fusion protein, called DysS9, bound to calmodulin-Sepharose, bound biotinylated calmodulin, caused characteristic changes in the fluorescence emission spectrum of dansyl-calmodulin, and had an apparent affinity for dansyl-calmodulin of 54 nM. Binding in each case was Ca2+-dependent. The maltose-binding protein does not bind calmodulin, and thus binding resides in the dystrophin-derived sequences. Deletion mutation experiments further localize the high affinity calmodulin binding to mouse dystrophin protein sequences 3293-3349, and this domain contains regions with chemical characteristics found in the calmodulin-binding sequences in other proteins. The COOH-terminal domain provides sites of attachment of dystrophin to membrane proteins, and calmodulin binding may modulate these interactions.

Novel DNA-Sepharose purification of the FadR transcription factor.

A DNA sequence bound by the FadR transcription factor of Escherichia coli was covalently attached to Sepharose by two different approaches: by chemical coupling or by template-directed enzymatic synthesis using a DNA polymerase. The two kinds of DNA-Sepharose were packed into small columns and used for the purification of the FadR protein; chromatography was without using competitor DNA and the supports contained single-copy, non-repetitive DNA sequences. Comparison showed that the enzymatically prepared support, while having less bound DNA, bound more FadR protein than did the chemically prepared support. This probably results from the lack of detrimental DNA modification by the gentle enzymatic procedure. The chemically prepared support was of lower capacity but yielded purer FadR protein when compared under the same elution conditions. This may be explained by the simpler DNA sequence which could be coupled chemically; less contaminating proteins were bound by the simpler DNA sequence. However, the enzymatically prepared support could also yield comparable purity if the protocol was modified to include additional washes with salt containing buffers. In all cases, FadR was eluted from the DNA using high-salt (0.8 M) mobile phase; ligand-specific elution of FadR using a fatty acyl-coenzyme A thiol ester was ineffective. Affinity chromatography on DNA-Sepharose provided a more rapid, simple purification of FadR than conventional purification techniques and yielded biologically active protein.

Alternative splicing dictates translational start in Epstein-Barr virus transcripts.

Of the 10 viral genes known to be expressed during Epstein-Barr virus (EBV) latency, six encode nuclear antigens (EBNAs), all of which are expressed from a long primary transcript by means of alternative splicing and alternative polyadenylation sites. The primary transcript is generated by either of two promoters which operate in a mutually exclusive fashion in different clonal cell lines. All mRNAs from either promoter have exons in common from the BamHI W viral genomic fragment (the major internal repeat, IR1) which encode the N-terminal portion of one of the nuclear antigens (EBNA 4). In addition to the coding regions for EBNA 4, EBNA mRNAs encode another EBNA (i.e. EBNA 1, 2, 3A, 3B or 3C) downstream. We show that alternative splicing determines whether the translation initiation codon for EBNA 4 is present or absent, thus permitting the generation of mRNAs in which the first translation initiation codon is either that for the EBNA 4 gene or for the other EBNA gene encoded downstream. This mechanism presumably ensures efficient translation of all the EBNA genes.

Bidirectional transcription of the Epstein-Barr virus major internal repeat.

In latently infected cell lines, the Epstein-Barr virus BamHI W fragment (major internal repeat) is transcribed in a rightward direction to yield exons common to several alternatively spliced messages which encode the six known viral nuclear antigens. A substantial steady-state population of very large (up to 20-kilobase) rightward transcripts is nuclear, much of it being polyadenylated. We report a rise in the levels of rightward transcripts hybridizing to BamHI-W sequences upon phorbol ester treatment of the clone-13 Burkitt's lymphoma cell line. We also report large (up to 15-kilobase) leftward transcripts hybridizing to BamHI-W sequences which occurred late in the viral lytic cycle in B95-8 and clone-13 cells. These leftward transcripts may antagonize the expression of the viral nuclear antigen messages by the formation of RNA duplexes.

Consumers Can Detect Light-Induced Flavor in Milk.

Two-thousand consumers were surveyed at three county fairs in Vermont to determine their taste preference for samples of good milk and milk with light-induced flavor defect. More than 73% of the people surveyed preferred the good milk sample. More females than males could taste a difference between the two samples, had a preference for one sample, and preferred the good sample. The data suggest strongly that it is in the best interests of the dairy industry to prevent light-induced flavor of milk.