Expression of two interleukin 4 mRNA isoforms in B lymphoid cells.

Expression of an alternative spliced IL4 mRNA was found in in vitro activated T cells. In this study we show that the expression of IL4 mRNA, as well as the expression of this alternatively spliced form of IL4 mRNA, is not restricted to these cells. We analyzed different human lymphoid tissues and cell lines of different origin and found that the alternatively spliced IL4 transcripts are also expressed in human lymphoid tissues, in purified B cells, in the various B cell-derived cell lines, and even in nonlymphoid cell lines. Stimulation with the phorbol ester PMA enhanced expression of both transcripts in all cells studied. The two IL4 transcripts were cloned and sequenced from the B cell line Namalwa. In the alternatively spliced form, the same exon 2 is deleted as has been observed in in vitro activated T cells. In principle, such alternatively spliced mRNA may give rise to a truncated IL4 protein, as the deletion does not result in a frameshift. We tested supernatants of activated PBMC, cell lines, and cell extracts for the presence of IL4 protein. We found IL4 protein expression in activated PBMC, but not in any of the stimulated cell lines or in the purified B cells. Using a modified in situ hybridization method with Dig-labeled PCR products, however, these cells did express IL4 mRNA. This shows that transcription of both IL4 forms is not restricted to T cells and can be induced in other cell types as well. Using these non-T cells, no protein has been found in the supernatant, however. It is possible that transcription of the IL4 gene is not necessarily followed by translation and that translation into the IL4 protein requires an additional signal.

Sequencing-based typing reveals new insight in HLA-DPA1 polymorphism.

An HLA-DPA1 sequencing-based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3' end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the--corrected--DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201.(ABSTRACT TRUNCATED AT 250 WORDS)

Asian Indian HLA-DR2-, DR4-, and DR52-related DR-DQ genotypes analyzed by polymerase chain reaction based nonradioactive oligonucleotide typing. Unique haplotypes and a novel DR4 subtype.

We have employed a PCR-based nonradioactive technique using biotinylated SSOPs to define HLA-DR2-, 4-, DR51-, and DR52-associated DR-DQ genotypes in Asian Indian families. In the DR2 group, most haplotypes described by us in a previous study were confirmed by family analysis. Evidence for one additional haplotype was available in this study. The classic DRB1*1501- and DRB1*1502-associated caucasoid haplotypes occurred with an appreciable frequency in Asian Indians, but two of the DRB1*1601-associated Caucasoid haplotypes were absent. At least six unique and unusual DR2-associated genotypes were encountered. In the DR52 group, the three most common alleles are DRB1*0301, DRB1*1404, and DRB1*1101. The DR6-associated alleles were DRB1*1301, 1302, 1401, and 1404. A few unique haplotypes occurred with low frequency in this group. In the DR4 group, at least three unusual patterns of hybridization were noticed by family analysis. One of these appears to be a novel DR4 subtype upon sequencing. These results demonstrate that, besides HLA-DR2, appreciable complexity occurs in the DR4- and DR52-associated alleles among Asian Indians. The presence of unique DR-DQ haplotypes in addition to those found characteristically among Western Caucasians suggests that the Indian population provides valuable source of many HLA class II haplotypes.

High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.

To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.

Assignment of HLA-DPB alleles by computerized matching based upon sequence data.

Routine HLA typing by serology has been supported by DNA or protein analysis of the respective molecules in cases when serologic typing was inconclusive or difficult to perform. DNA analysis by RFLP, SSO, or PCR amplification with SSP is a reliable tool for the identification of alleles. Because DNA sequences may now be determined routinely, we developed software based on sequence data from known sequences for allele assignment of polymorphic gene systems. We describe the assignment of HLA-DPB alleles based upon sequence data obtained after PCR amplification and subsequent sequencing of exon 2. Our software includes a database containing all known HLA-DPB sequences, which offers the possibility of analyzing heterozygous individuals by combinatorial comparison through all alleles and thus identifying the one or two alleles involved. Quality control of the sequence has been included by the alignment of constant regions of the sequence combined with related polymorphism of known polymorphic nucleotides and the identification of the positions crucial for the allele assignment. The ability to type for HLA-DPB alleles routinely by automatic sequence determination and subsequent automated analysis offers new perspectives for HLA-DNA typing.

Aberrant oestrogen receptor species in human meningioma tissue.

Meningiomas are very rich in progestin receptors (PR) whereas oestrogen receptors (ER) are seldomly found and only at low concentrations. These tumours might possess an ER which is defective in oestrogen binding but still functional in stimulating oestrogen-responsive genes such as PR. In human meningiomas a polymerase chain reaction fragment including the DNA binding domain, the hinge region and the ligand binding domain of ER was amplified. The size of the fragment obtained was as expected from wild type mRNA sequences. Moreover, a variant, which was overexpressed in meningiomas, with a major deletion in exons 2-6 was detected.