Summary of validation studies from twenty-six forensic laboratories in the United States and Canada on the use of the AmpliType PM PCR amplification and typing kit.

A cooperative study was undertaken to collect and summarize the results of validation studies from forensic laboratories in the United States and Canada on the use of the AmpliType PM PCR amplification and typing kit for genetic typing of forensic biological evidence. This report compiles data from 26 laboratories on: 1) reproducibility studies on DNA extracted from various samples, 2) genetic typing of DNA extracted from a variety of biological samples on various substrates, 3) the effects of exogenous chemicals, materials, and environmental factors on test results, 4) sensitivity studies to determine the least detectable amount of extracted genomic DNA that can be reliably typed, 5) analysis of mixtures containing two sources of genomic DNA, 6) cross-hybridization with DNA extracted from various nonhuman species, and 7) evaluation of assay performance on parallel studies with other genetic typing systems on proficiency test panels, mock cases, and adjudicated/nonprobative casework. Equivalent results were obtained by each laboratory that supplied data, demonstrating the reliability and consistency of the test. Overall, it can be concluded from this study that the AmpliType PM PCR amplification and typing kit meets the guidelines of the Technical Working Group on DNA Analysis Methods (TWGDAM) and there is general scientific acceptability of this kit for forensic DNA testing.

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.

Immunoglobulin D switching can occur through homologous recombination in human B cells.

Prototypical class switching in mouse and human immunoglobulin heavy chains occurs through recombination of tandem blocks of short repeats located 5' to each heavy chain constant region (CH) except C delta. Deletion of C mu in immunoglobulin D (IgD)-secreting murine plasmacytomas occurs illegitimately. We demonstrate here that in human IgD-secreting myeloma cells freshly isolated from patient bone marrow and in normal peripheral blood B lymphocytes, an IgD switch can occur through homologous recombination of a direct repeat consisting of a 442-bp sequence 1.5 kbp 3' of the JH complex and a 443-bp sequence that is duplicated almost perfectly (96% similarity) 1.7 kbp 5' of the C delta gene (442/443-base-pair [bp] repeat). This homologous recombination mechanism is not exclusive for IgD switching, since C mu deletion endpoints in two established IgD-secreting myeloma cell lines fall outside the 442/443-bp repeat. The 442/443-bp mediated recombination shows cell type specificity, and we propose that it represents a unique mode for increased levels of IgD secretion in humans.

Antisera to the secretory component recognize the murine Fc receptor for IgA.

Studies were undertaken to determine a possible structural relationship between the secretory component (SC) and the receptor for IgA (Fc alpha R). An IgA-mediated rosetting technique was used to assess the presence of Fc alpha R+ cells in various lymphoid tissues from normal BALB/c mice and mice bearing an IgA plasmacytoma (MOPC 315). Tissues from the MOPC 315-bearing BALB/c mice were found to have a significantly higher percentage of Fc alpha R+ cells; thus, nonadherent spleen cells from MOPC 315-bearing mice were used as a source of Fc alpha R+ cells in these studies. The cells were preincubated with anti-SC and then assayed for the ability of IgA to bind to the Fc alpha R. Antisera to SC from various species inhibited the formation of IgA-mediated rosettes, although preincubation of the Fc alpha R+ cells with antisera directed against other cell surface molecules (e.g., Thy1.2, Lyt1, Lyt2, Fc gamma R, MHC class I and II) or preimmune sera had no significant effect on IgA-mediated rosette formation. Preabsorption of the anti-SC with secretory IgA or with free SC removed the inhibitory effect; preabsorption with myeloma IgA had no effect. These data suggest that SC and Fc alpha R are related serologically and may be structurally related, possible in the IgA-binding region.

The human immunoglobulin C mu-C delta locus: complete nucleotide sequence and structural analysis.

The entire nucleotide sequence (approximately 20 kbp) spanning the human immunoglobulin IgM (mu) and IgD (delta) heavy chain constant region genes has been determined from DNA of mu-delta producing chronic lymphocytic leukemic B cells. As in the murine IgM + IgD double-producing B cells, no rearrangement has occurred in the C mu-C delta region in the leukemic cells. The C mu locus is highly conserved between mouse and human with the exception of the nucleotide sequence between the C mu 4 and mu M1 exons, which has diverged dramatically. The intergenic sequence between human C mu and C delta is three times larger than the analogous region in the mouse and contains notable features absent from the mouse, including a 443 bp segment that is 96% identical to a 442 bp sequence that occurs just 3' to the heavy chain enhancer, a 366 bp sequence that is directly repeated with 76% homology, and 12 tandem copies of a 35 bp sequence. The human C delta gene contains two additional exons relative to mouse C delta, but shares with the mouse the unique distal location of both secreted and membrane coding segments. Several polymorphisms in the human population have been identified in the intergenic region and in C delta but not in C mu.

The human immunoglobulin C mu-C delta locus: regulation of mu and delta RNA expression during B cell development.

Whether the immunoglobulin (Ig) heavy chain genes C mu and C delta are expressed singly or in combination, their transcripts undergo differentiation-specific alterations in membrane (M) versus secreted (S) forms as well as in abundance. To better understand this regulation, we have cloned cDNAs for human delta m and delta s to establish the 3' end of the C mu-C delta transcription unit. Steady state mRNA levels and transcription rates were then analyzed in normal and transformed human B cells representing different maturation and activation states. The ratio of micron/microsecond RNA and of delta m/delta s RNA correlated with developmental stage, with a higher ratio at earlier stages. Steady state ratios of total mu/delta RNA paralleled ratios of C mu/C delta nascent transcription, suggesting no major posttranscriptional control for differential expression. However, at all developmental stages, transcription termination occurred downstream of the micron exons, suggesting a strong posttranscriptional regulatory component for production of secreted versus membrane forms of mu RNA. The relative abundance of mature delta S RNA was considerably higher in the human than in the mouse, correlating with the increased levels of circulating IgD in the former species. Stimulation of human splenocytes with mitogens did not increase delta RNA; in fact, splenocytes activated with pokeweed mitogen were nearly devoid of delta RNA, and Staphylococcus aureus Cowan I caused only a minor change.

Double isotype production by a neoplastic B cell line. I. Cellular and biochemical characterization of a variant of BCL1 that expresses and secretes both IgM and IgG1.

We have subcloned the in vitro-adapted murine B cell leukemia, BCL1.B1, to obtain a variant that expresses both IgM and IgG1. By fluorescence analysis, radioiodination, and immunoprecipitation of cell surface Ig, and by RIA of medium from limiting dilution cultures, we have shown that: (a) all the cells express and secrete both isotypes. The heavy chains of both IgG1 and IgM have the apparent molecular weights of membrane mu and gamma 1 chains; (b) both isotypes bear the same idiotype as determined by immunoprecipitation with antiidiotypic antibody, and both use the same VDJ rearrangement as shown by Southern blotting; and (c) the cells express the membrane and secreted forms of mRNA for both mu and gamma 1 but not gamma 2b or gamma 3. Taken together, the data suggest that all the cells are synthesizing, expressing on their surface, and secreting two isotypes that use the same VDJ rearrangement in the DNA and express the same serologically-defined idiotype. The molecular basis responsible for the production of the two isotypes in a single cell is the subject of the accompanying paper.

Regulation of IgA expression by isotype-specific T cells and soluble binding factors.

The data presented here suggest a model for isotype-specific regulation of IgA synthesis by Fc alpha R+ T cells (Figure 1). Immature mIgM+ +/- mIgD+ B cells are induced by T switch cells to express cell surface IgA (a phenotypic switch). If the T switch cell induces mIgA expression via a long primary RNA transcript from an unrearranged C alpha allele, the hypothetical intermediate switch B cell results (step 1); this may be the mechanism of heavy chain expression in memory B cells that express low levels of Ig. Alternatively, T switch cells may induce a DNA rearrangement in the CH locus of the B cell (a genotypic switch), which results in a deletion of all CH loci except C alpha (step 2). TH inducer cells promote maturation of mIgA+ B cells to IgA-secreting plasma cells. This may involve a DNA switch rearrangement (step 3) or the maturation of previously switched cells (step 4), and appears to be mediated via an IgABF with enhancing activity. Not shown in this figure, but inherent in this model, is a suppressive regulatory arm that may be mediated via IgABF with suppressive activity released from Fc alpha R+ suppressor T cells. Due to the presence of Fc alpha R on a variety of cell types, IgABF may suppress synthesis of IgA by acting not only on mIgA+ B cells but also on regulatory cells (T cells, B cells, and macrophages) bearing IgA bound to Fc alpha R. If the IgA system is analogous with the IgE system, mIgA-bearing B cells may be the direct target of IgABF. Binding of Ig to FcR has been shown to (a) increase the number of Fc receptors per cell, (b) enhance the number of cells expressing Fc receptors, (c) induce the release of IgBF that either suppress or enhance Ig secretion, and (d) effectively convert surface Ig- cells into surface Ig+ cells that are therefore receptive to IgBF. Thus, FcR+ cells may interact with IgBF and Ig via a regulatory network to stimulate or inhibit the immune response in an isotype-specific manner. Cell surface molecules (mIg, FcR) may serve as sensors that allow the cell to detect and respond to fluctuations in the levels of immune mediators that serve to modulate Ig synthesis and secretion. The relationship between IgBF and FcR is not known, nor is it known whether Fc receptors expressed by different cell types are encoded by the same gene and are controlled similarly.(ABSTRACT TRUNCATED AT 400 WORDS)

Human immunoglobulin D: genomic sequence of the delta heavy chain.

The DNA coding for the human immunoglobulin D(IgD) heavy chain (delta, delta) has been sequenced including the membrane and secreted termini. Human delta, like that of the mouse, has a separate exon for the carboxyl terminus of the secreted form. This feature of human and mouse IgD distinguishes it from all other immunoglobulins regardless of species or class. The human gene is different from that of the mouse; it has three, rather than two, constant region domains; and its lengthy hinge is encoded by two exons rather than one. Except for the third constant region, the human and mouse genes are only distantly related.

Structural analysis of the murine IgG3 constant region gene.

We have determined the complete sequence of the gamma 3 heavy chain constant (C gamma 3) region gene of the BALB/c mouse including the 5'-flanking region up to the switch site and the 3'-flanking region past the end of the membrane exons. The C gamma 3 coding region, typical of other IgGs, is divided into six exons corresponding to the protein domains (C gamma (3)1, hinge, C gamma (3)2, and C gamma (3)3) and to the membrane carboxyl terminus (M1 and M2). The predicted amino acid sequence of the gamma 3 chain has three potential N-linked carbohydrate addition sites (including one in the membrane spacer segment), as compared with a single occurrence in the other mouse IgGs. Between the switch recombination region and the body of the C gamma 3 gene, there is a remarkable homology with a sequence between C mu and C delta which provides a rationale for an alternative, T cell-independent class-switch mechanism. We have used a computer to analyze the secondary structure of the gamma 3 mRNA precursor for the membrane form. We predict that this RNA precursor (approximately 12 000 bp) folds into four leaf-like domains which correspond to the variable region, the large IVS, the body of the constant region, and the membrane exons. This organization may have a role to play in the function of the mRNA precursor.

The murine immunoglobulin alpha gene expresses multiple transcripts from a unique membrane exon.

Immunoglobulin heavy chains of each class exist in two forms, membrane (m) and secreted (s), which have different carboxy-terminal sequences encoded by different gene segments. We have sequenced the DNA spanning the coding region for the carboxy-terminal end of the alpha m chain in a BALB/c genomic clone and have examined alpha mRNA expression in transformed and normal B cells. Unlike the other murine heavy chain genes, which have two membrane exons (M1 and M2), the alpha gene has only one membrane exon (alpha M1). This unusual exon encodes a highly conserved transmembranal peptide plus flanking amino acids singular to IgA. The complexity of the alpha gene transcription is also unusual. Downstream of alpha M1, three AATAAA hexanucleotides are used to terminate as many as six distinct alpha membrane mRNAs. These appear to differ only in length and splicing pattern of their 3'-untranslated regions, and thus, encode the same alpha m protein. The alpha s protein is approximately 4000 daltons smaller than alpha m as judged by in vitro translation and is coded by a single 2.0-kb alpha s mRNA. We propose that the expression of alpha s and the multiple alpha m mRNAs is differentially controlled at the level of transcriptional termination.