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An approach for generating DNA profiles when critical samples have been consumed and a power outage occurs during the polymerase chain reaction (PCR) amplification reaction is described. This study demonstrates that a complete and accurate DNA short tandem repeat profile can be obtained: (1) when single source DNA samples are amplified for 26, 27, or 28 cycles using the Profiler Plus and COfiler Amplification Kits after an interruption in amplification, (2) from mock samples when PCR amplification has been interrupted early (after five cycles) or late (after 18 cycles) and the sample is subjected to an additional round of amplification, even after incubation of the sample at room temperature overnight, and (3) from nonprobative casework samples interrupted after approximately 18 cycles of amplification, an overnight incubation at room temperature and subjected to one or two additional rounds of PCR amplification for approximately 26 total cycles. Samples interrupted before five completed cycles and subjected to additional PCR cycles yielded variable results.

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.

This primer is a tool to help prepare scientists for meetings with stakeholders. It was prepared for staff involved with the Natural and Accelerated Bioremediation Research (NABIR) program, sponsored by the U.S. Department of Energy. It discusses why some efforts in science communication may succeed while others fail, provides methods of approaching group interactions about science that may better orient expert participants, and summarizes experience drawn from observations of @oups interacting about topics in bioremediation or the NABIR program. The primer also provides briez usefid models for interacting with either expert or non-expert groups. Finally, it identifies topical areas that may help scientists prepare for public meetings, based on the developers' ongoing research in science communication in public forums.

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.

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.

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.

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.

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 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)

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.

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.