DNA data banking: a cautionary tale.
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A review of state legislation on DNA forensic data banking.
Recent advances in DNA identification technology are making their way into the criminal law. States across the country are enacting legislation to create repositories for the storage both of DNA samples collected from convicted offenders and of the DNA profiles derived from them. These data banks will be used to assist in the resolution of future crimes. This study surveys existing state statues, pending legislation, and administrative regulations that govern these DNA forensic data banks. We critically analyzed these laws with respect to their treatment of the collection, storage, analysis, retrieval, and use of DNA and DNA data. We found much variation among data-banking laws and conclude that, while DNA forensic data banking carries tremendous potential for law enforcement, many states, in their rush to create data banks, have paid little attention to issues of quality control, quality assurance, and privacy. In addition, the sweep of some laws is unnecessarily broad. Legislative modifications are needed in many states to better safeguard civil liberties and individual privacy.
Advisory Committees↗
Inhibition of parvovirus minute virus of mice replication by a peptide involved in the oligomerization of nonstructural protein NS1.
The large nonstructural protein NS1 of the minute virus of mice and other parvoviruses is involved in essential steps of the viral life cycle, such as DNA replication and transcriptional regulation, and is a major contributor to the toxic effect on host cells. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to NS1. Homo-oligomerization is a prerequisite for a number of proteins to be fully functional. In particular, helicases generally act as homo-oligomers. Indirect evidence of NS1 self-association has been recently obtained by a nuclear cotransport assay (J. P. Nüesch and P. Tattersall, Virology 196:637-651, 1993). In order to demonstrate the oligomerizing property of NS1 in a direct way and localize the protein region(s) involved, the yeast two-hybrid system was used in combination with deletion mutagenesis across the whole NS1 molecule, followed by high-resolution mapping of the homo-oligomerization domain by a peptide enzyme-linked immunosorbent assay method. This study led to the identification of a distinct NS1 peptide that contains a bipartite domain involved in NS1 oligomerization. Furthermore, this isolated peptide was found to act as a specific competitive inhibitor and suppress NS1 helicase activity in vitro and parvovirus DNA replication in vivo, arguing for the involvement of NS1 oligomerization in these processes. Our results point to drug targeting of oligomerization motifs of viral regulatory proteins as a potentially useful antiviral strategy.
Amino Acid Sequence↗
The structural complexities of the myelin basic protein gene from mouse are also present in shark.
The Golli-mbp gene complex contains two overlapping transcription units with two distinct promoters, of which the downstream (myelin basic protein [mbp]) promoter is more frequently used. A previous comparison of the downstream promoter sequences from shark and mouse allowed the identification of two DNA sequences called the boxes I and II and the wobble zone. The boxes I and II sequence is a composite cis-acting motif that is thought to be involved in the regulation of the downstream promoter. It contains sequences similar to T-antigen, MyoD/E2A, and glucocorticoid receptor-binding sites. The wobble zone codes for an exon (5a in the nomenclature of Campagnoni et al., 1993) that is included in messenger RNAs transcribed from the upstream promoter. The polypeptides encoded by this exon from shark and mouse are 86 and 84 amino acids long, respectively. These polypeptides are overall 59% identical and include a region (residues 41-75 in shark and 39-73 in mouse) that is 89% identical between the two species. A primary sequence analysis showed that each of these polypeptides contains an N-glycosylation site, phosphorylation sites for Ca2+/calmodulin-dependent protein kinase, protein kinase C and casein kinase II, and partial ATP- and GTP-binding sites. The shark polypeptide also contains a phosphorylation site for proline-directed protein kinase. These observations are consistent with the notion that the intricate structure and regulation of the Golli-mbp gene complex arose during vertebrate evolution within a common ancestor to sharks and mammals.
Amino Acid Sequence↗
Functional mapping of regions of the Autographa californica nuclear polyhedrosis viral genome required for DNA replication.
Previous results showed that plasmids containing one of the eight putative origins (ori's) of Autographa californica nuclear polyhedrosis virus (AcMNPV) are replicated after transfection into Spodoptera frugiperda cells if essential trans-acting factors are supplied by AcMNPV infection (Kool et al., Virology, 192, 94-101, 1993a; Kool et al., J. Gen. Virol., in press, 1993b; Leisy and Rohrmann, Virology, 196, 722-730, 1993). In this report a transient complementation assay is described in which four cotransfected cosmid clones, instead of AcMNPV infection, provided essential trans-acting factors for plasmid DNA replication. In this assay plasmid replication was found to be independent of the presence, in cis, of a viral ori. No replication of plasmids occurred when one of the cosmids was omitted from the transfection mixture. This result indicated that this assay is a valid approach for identification of AcMNPV replication genes. We further used the assay to define essential regions in the four required cosmids. Six regions of the AcMNPV genome, EcoRI-I (map unit 0.3-5.8), EcoRI-O (map unit 6.9-8.7), SstI-F (map unit 38.9-45.0), EcoRI-D (map unit 59.9-68.3), a BamHI-SstII fragment of BamHI-B (map unit 84.3-89.7), and EcoRI-B (map unit 90.0-100), with at least seven genes, were found to be essential for plasmid DNA replication. These regions contain the putative DNA polymerase gene (SstI-F), the helicase-like gene (EcoRI-D), and the region where most of the trans-activating immediate--early genes of AcMNPV are located (EcoRI-B). For SstI-F it was shown that this region contains besides the DNA polymerase gene at least one other replication gene. These results show that it will now be possible to define the set of AcMNPV genes necessary and sufficient for DNA replication.
Animals↗
Transcription factors RFX1/EF-C and ATF-1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter.
The proliferating cell nuclear antigen (PCNA) is an adenovirus E1A-inducible factor that is intimately linked to the processes of DNA replication and cell cycle regulation. Previously, we defined a novel cis-acting element, the PCNA E1A-responsive element (PERE), that confers induction by the E1A 243R oncoprotein upon the human PCNA promoter. To better understand the regulation of PCNA expression by E1A 243R, we have identified cellular transcription factors that associate with the PERE. In electrophoretic mobility shift assays, the PERE formed three major complexes (P1, P2 and P3) with proteins in nuclear extracts from HeLa or 293 cells. Formation of complexes P2 and P3, which correlates with PCNA promoter activity in vivo, requires the activating transcription factor (ATF) binding site found within the PERE [Labrie et al. (1993) Mol. Cell. Biol., 13, 1697-1707]. Antibody interference experiments and mobility shift assays performed with in vitro-synthesized protein indicated that the transcription factor ATF-1 is a major component of these complexes. Similar assays demonstrated that the hepatitis B virus enhancer-associated protein RFX1 constitutes a major component of the P1 complex. In addition, we examined the binding of proteins to the minimal E1A-responsive promoter to identify other factors important for transcription from the PCNA promoter. Mobility shift assays revealed that a fragment encompassing the region from -87 to +62 relative to the transcription initiation site forms at least five complexes, EH1-EH5, with HeLa cell nuclear extracts. The transcription factor YY1 associates with the initiator element of the PCNA promoter. The identification of these transcription factors will allow their roles in the activation of PCNA by E1A to be evaluated.
Activating Transcription Factor 1↗
The identification of rofA, a positive-acting regulatory component of prtF expression: use of an m gamma delta-based shuttle mutagenesis strategy in Streptococcus pyogenes.
Binding of the Gram-positive pathogenic bacterium Streptococcus pyogenes (group A streptococcus) to respiratory epithelium is mediated by the fibronectin-binding adhesin, protein F. Most strains of streptococci regulate the expression of protein F in response to oxygen levels and redox potential; however, JRS4 constitutively binds high levels of fibronectin under all environmental conditions. In this study, we have examined the regulation of protein F expression in JRS4 using a shuttle mutagenesis strategy novel to S. pyogenes. Cloned DNA representing the chromosomal loci adjacent to the gene which encodes protein F (prtF) was subjected to transposon mutagenesis in Escherichia coli using a derivative of transposon m gamma delta that was modified to contain a streptococcal antibiotic-resistance gene. mutagenized DNA was then returned to the streptococcal chromosome by allelic replacement. Analysis of the resulting fibronectin-binding phenotypes revealed that insertions in a region upstream of prtF abolished the constitutive phenotype. However, these mutants now demonstrated regulation in response to both oxygen levels and redox potential. Because these insertions define a locus responsible for the constitutive phenotype, it has been designated rofA (regulator of F). Chromosomal interruption studies using integrational plasmids together with complementation data from a previous study (VanHeyningen et al., 1993) suggested that rofA acts as a positive trans-acting regulator of prtF. Construction of prtF-lacZ fusions indicated that transcription of prtF is constitutive in JRS4 but is regulated in rofA mutants. Analysis of the DNA sequence defined by the rofA insertions revealed a 1495 bp open reading frame, whose predicted product (RofA) possessed both a putative helix-turn-helix motif and limited homology to two other transcriptional activators (Mry, PrgR) of Gram-positive surface proteins. Sequences homologous to rofA were found in regulated strains of S. pyogenes, which suggests that rofA may act as an activator of prtF in response to an unidentified environmental signal. We speculate that the allele reported here contains a mutation that renders it constitutively active.
Amino Acid Sequence↗
Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene.
Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.
Amino Acid Sequence↗
Advanced maternal age and the risk of Down syndrome characterized by the meiotic stage of chromosomal error: a population-based study.
The identification of DNA polymorphisms makes it possible to classify trisomy 21 according to the parental origin and stage (meiosis I [MI], meiosis II [MII], or postzygotic mitotic) of the chromosomal error. Studying the effect of parental age on these subgroups could shed light on parental exposures and their timing. From 1989 through 1993, 170 infants with trisomy 21 and 267 randomly selected control infants were ascertained in a population-based, case-control study in metropolitan Atlanta. Blood samples for genetic studies were obtained from case infants and their parents. Using logistic regression, we independently examined the association between maternal and paternal age and subgroups of trisomy 21 defined by parental origin and meiotic stage. The distribution of trisomy 21 by origin was 86% maternal (75% MI and 25% MII), 9% paternal (50% MI and 50% MII), and 5% mitotic. Compared with women <25 years of age, women > or = 40 years old had an odds ratio of 5.2 (95% confidence interval, 1.0-27.4) for maternal MI (MMI) errors and 51.4 (95% confidence interval, 2.3-999.0) for maternal MII (MMII) errors. Birth-prevalence rates for women > or = 40 years old were 4.2/1000 births for MMI errors and 1.9/1000 for MMII errors. These results support an association between advanced maternal age and both MMI and MMII errors. The association with MI does not pinpoint the timing of the error; however, the association with MII implies that there is at least one maternal-age related mechanism acting around the time of conception.
Adult↗
Growth phase-dependent regulation and membrane localization of SpaB, a protein involved in biosynthesis of the lantibiotic subtilin.
The information responsible for biosynthesis of the lantibiotic subtilin is organized in an operon-like structure that starts with the spaB gene. The spaB gene encodes an open reading frame consisting of 1,030 amino acid residues, and it was calculated that a protein having a theoretical molecular mass of 120.5 kDa could be produced from this gene. This is consistent with the apparent molecular weight for SpaB of 115,000 which was estimated after sodium dodecyl sulfate-gel electrophoresis and identification with SpaB-specific antibodies. The SpaB protein is very similar to proteins EpiB and NisB, which were identified previously as being involved in epidermin and nisin biosynthesis. Upstream from SpaB a characteristic sigma A promoter sequence was identified. An immunoblot analysis revealed that SpaB expression was strongly regulated. No SpaB protein was detected in the early logarithmic growth phase, and maximum SpaB expression was observed in the early stationary growth phase. The expression of SpaB was strongly correlated with subtilin biosynthesis. Deletion mutations in either of two recently identified regulatory genes, spaR and spaK, which act as a "two-component" regulatory system necessary for growth phase-dependent induction of subtilin biosynthesis (C. Klein, C. Kaletta, and K. D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), also resulted in failure of SpaB expression. To investigate the intracellular localization of SpaB, vesicles of Bacillus subtilis were prepared. The SpaB protein cosedimented with the vesicle fraction and was released only after vigorous resuspension of the vesicles. Our results suggest that SpaB is membrane associated and that subtilin biosynthesis occurs at the cytoplasmic membrane of B. subtilis.
Amino Acid Sequence↗
Re-evaluation of primary structure, topology, and localization of Scamper, a putative intracellular Ca2+ channel activated by sphingosylphosphocholine.
Naturally occurring sphingoid molecules control vital functions of the cell through their interaction with specific receptors. Proliferation, differentiation and programmed death result in fact from a fine balance of signals, among which sphingosine and structurally related molecules play fundamental roles, acting as either first or second messengers. The corresponding receptors need to be identified in order that the role of sphingoid molecules can be established. Among them, several G-protein-coupled receptors specific for sphingosine 1-phosphate, sphingosylphosphocholine, or both, have already been investigated. In contrast, the identification of the postulated intracellular receptors has been problematical. In the present study we re-evaluated the molecular characterization of Scamper, the first proposed intracellular receptor for sphingosylphosphocholine [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1993-1996] and commonly believed to be a Ca(2+) channel of the endoplasmic reticulum (the name "SCaMPER" used by Mao et al. being derived from "sphingolipid Ca(2+)-release-mediating protein of the endoplasmic reticulum"). In contrast with what has been believed hitherto, our primary-structure and overexpression experiments indicate that Scamper is a 110-amino-acid protein spanning the membrane once with a Nexo/Ccyt topology [von Heijne and Gavel (1988) Eur. J. Biochem. 174, 671-678]. Overexpression of either wild-type or tagged Scamper induces a specific phenotype characterized by the rapid extension of actin-containing protrusions, followed by cell death.
Amino Acid Sequence↗
Identification of a negative cis-regulatory element and trans-acting protein that inhibit transcription of the angiotensin II type 1a receptor gene.
The rat angiotensin II type 1a receptor (AT1a-R) gene is expressed in a cell-specific manner. We demonstrated that the negative regulatory element (NRE) between -489 and -331 is active in PC12 cells (Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003). Gel retardation assays confirmed that PC12 cells have a trans-acting factor bound to the NRE. By means of a DNase I footprint assay we identified the core of the NRE as an (A+T)-rich sequence (TAATCTTTTATTTTA) located at nucleotides -456 to -442. Oligonucleotides corresponding to the NRE core sequence bound to nuclear protein. Site-directed mutagenesis at nucleotides -451 to -448 eliminated the specific protein/DNA binding and restored expression of the AT1a-R in transient transfection assays (2.7-fold increase). The NRE did not negatively affect the thymidine kinase promoter. No homology was found with known NREs, suggesting that this is a novel NRE. Southwestern blotting revealed a 53-kDa, specific binding protein in PC12 cells and the rat brain, but not in the liver, spleen, adrenal gland, and kidney. These findings demonstrate that the NRE of the rat AT1a-R is an (A+T)-rich sequence located at nucleotides -456 to -442 and the 53-kDa protein is a specific binding protein, and suggest that this protein may be a trans-acting factor which determines the neuron-specific down-regulation of the AT1a-R gene.
Angiotensin II↗
Cleavage of RNA hairpins mediated by a developmentally regulated CCCH zinc finger protein.
Control of RNA turnover is a major, but poorly understood, aspect of gene regulation. In multicellular organisms, progress toward dissecting RNA turnover pathways has been made by defining some cis-acting sequences that function as either regulatory or cleavage targets (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993). However, the identification of genes encoding proteins that regulate or cleave target RNAs has been elusive (C. A. Beelman and R. Parker, Cell 81:79-183, 1995); this gap in knowledge has made it difficult to identify additional components of RNA turnover pathways. We have utilized a modified expression cloning strategy to identify a developmentally regulated gene from Drosophila melanogaster that encodes a RNase that we refer to as Clipper (CLP). Significant sequence matches to open reading frames encoding unknown functions identified from the Caenorhabditis elegans and Saccharomyces cerevisiae genome sequencing projects suggest that all three proteins are members of a new protein family conserved from lower eukaryotes to invertebrates. We demonstrate that a member of this new protein family specifically cleaves RNA hairpins and that this activity resides in a region containing five copies of a previously uncharacterized CCCH zinc finger motif. CLP's endoribonucleolytic activity is distinct from that associated with RNase A (P. Blackburn and S. Moore, p. 317-433, in P. D. Boyer, ed., The Enzymes, vol. XV, part B, 1982) and is unrelated to RNase III processing of rRNAs and tRNAs (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993, and S. A. Elela, H. Igel, and M. Ares, Cell 85:115-124, 1995). Our results suggest that CLP may function directly in RNA metabolism.
Amino Acid Sequence↗
Enhancer-specific modulation of E protein activity.
Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes (Bain, G., Gruenwald, S., and Murre, C. (1993) Mol. Cell Biol. 13, 3522-3529; Shen, C. P., and Kadesch, T. (1995) Mol. Cell Biol. 15, 4518-4524; Jacobs, Y., et al. (1994) Mol. Cell Biol. 14, 4087-4096; Wilson, R. B., et al. (1991) Mol. Cell Biol. 11, 6185-6191). These homodimers, however, have little activity on myogenic enhancers (Weintraub, H., Genetta, T., and Kadesch, T. (1994) Genes Dev. 8, 2203-2211). We report here the identification of a novel cis-acting transcriptional repression domain in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, we demonstrate that Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes.
Amino Acid Sequence↗