Interleukin-4 receptor expression by human B cells: functional analysis with a human interleukin-4 toxin, DAB389IL-4.

BACKGROUND: Studies of human IgE-secreting B cells have proven difficult because of the small size of this population. We have used an interleukin-4 (IL-4) fusion toxin to detect functionally IL-4 receptor (IL-4R) expression on B cells involved in IgE synthesis. METHODS: In diphtheria toxin IL-4 (DAB389IL-4) the receptor-binding domain of diphtheria toxin has been replaced with human IL-4. DAB389IL-4 cytotoxicity depends on IL-4R binding and internalization. RESULTS: Addition of DAB389IL-4 inhibited IgE synthesis induced by IL-4+ anti-CD40 monoclonal antibody or hydrocortisone. IgE inhibition resulted from DAB389IL-4 B-cell cytotoxicity because DAB389IL-4 inhibited IL-4-independent B-cell proliferation. Thus induction of human IgE synthesis involves IL-4R+ cells. In contrast, terminally differentiated, IgE-producing B cells no longer express functional IL-4R because DAB389IL-4 only modestly inhibited ongoing IgE synthesis by B cells from patients with hyper-IgE states and only minimally affected IL-4-induced IgE synthesis in normal B cells when the toxin was added at day 7. Pokeweed mitogen-induced IgM synthesis was sensitive to early but not to late addition of DAB389IL-4. Thus the loss of functional IL-4R immunoglobulin-secreting B cells is independent of isotype switching. CONCLUSIONS: IgE-secreting B cells no longer express functional IL-4R. Therapies for allergic disease that target the IL-4R would not affect IgE-secreting B cells but may block the recruitment of B cells into the IgE-secreting pool. For optimal benefits this approach may be combined with therapies that target IL-4R-, IgE-secreting B cells.

Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins.

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.

The urea amidolyase (DUR1,2) gene of Saccharomyces cerevisiae.

The DNA sequence of the urea amidolyase (DUR1,2) gene from S. cerevisiae has been determined. The polypeptide structure deduced from the DNA sequence contains 1,835 amino acid residues and possesses a calculated weight of 201,665 daltons which favorably correlates with that predicted from compositional analysis of purified protein (1,881 amino acid residues and a molecular weight of 203,900). The C-terminal 57 residues of the polypeptide exhibit significant homology with similarly situated sequences found in five other biotin carboxylases whose primary structures have been determined or deduced from protein and DNA sequence data, respectively. Major S1 nuclease protection fragments derived from DUR1,2 RNA-DNA hybrids exhibit apparent termini at positions -140 and -141 upstream of the coding region. The termini of minor protection fragments also occur at eleven other positions as well.

Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae.

DAL5 is a constitutively expressed allantoin system gene whose product is required for allantoate transport. Its simple pattern of expression prompted us to use this gene for identifying the element(s) that mediates transcriptional activation of allantoin system genes. Deletion analysis of the DAL5 5'-flanking sequences resulted in identification of two small regions required for DAL5 expression. Analysis of these two regions with synthetic oligonucleotides localized the sequences supporting transcriptional activation to two DNA fragments of 10 to 12 base pairs, each containing one copy of the pentanucleotide 5'-GATAA-3'. The 5'-flanking region of DAL5 contained eight copies of this sequence. Synthetic constructions containing single copies of these fragments were unable to support transcriptional activation, while those containing two or more copies supported high-level activation. The 5'-GATAA-3' sequence was also found beneath the footprint of a DNA-binding protein. These observations are consistent with the suggestion that DNA fragments containing the sequence 5'-GATAA-3' play an important role in DAL5 gene expression, probably representing a portion of the binding site for a transcriptional activation factor.

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

Structure and transcription of the allantoate permease gene (DAL5) from Saccharomyces cerevisiae.

We determined the nucleotide sequence of the DAL5 gene, which encodes a component of the allantoate transport system. Translation of the sequence revealed that the DAL5 gene product is highly hydrophobic. It possesses an alternating motif of hydrophilic sequences that can potentially be folded into alpha-helices and hydrophobic sequences that can potentially be folded into beta-pleated sheets. These are expected characteristics of an integral membrane protein, which correlate well with DAL5 gene function. S1 protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range. This pattern of fragments was not affected by growth conditions of the cells or the conditions of the assay.

Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae.

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.

Tau, sigma, and delta. A family of repeated elements in yeast.

We report here the isolation and structure of a new repeated DNA element, tau. This element, from Saccharomyces cerevisiae, is 371 base pairs long and is flanked on either end by the same invertedly repeated sequence found at the ends of some Ty and sigma elements in yeast, copia elements in Drosophila and spleen necrosis virus. The tau inverted repeats are themselves flanked by a 5-base pair directly repeated genomic sequence that is present only once in a cognate tau-allele. These structural characteristics, the presence of multiple copies of tau in the genome, and the isolation of tau+ and tau- allelic pairs suggest that tau may be capable of transposition either alone or in association with some larger element. Detailed sequence analysis of the tau, sigma, and delta elements revealed that all three contain significant regions of homology, suggesting that they are probably members of a single family derived from a common progenitor.

tau, a repeated DNA sequence in yeast.

We have found a 371-base-pair (bp) repeated DNA element, tau, in Saccharomyces cerevisiae. The ends of tau are composed of a 5-bp inverted repeat, similar in sequence to those reported for the Ty, sigma, copia, and spleen necrosis virus elements. These inverted repeats are flanked by 5-bp direct repeats of a target sequence that occurs only once in an allele that lacks the tau element. This overall structure is characteristic of transposable elements. Like sigma, tau elements have been found (in both orientations) closely associated with tRNA genes (409 and 198 bp from the 5' end, respectively). It is noteworthy that one representative of tau was isolated in a concentric insertion of tau, delta, and sigma.