The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Quantitation of Human Metallothionein Isoforms: A Family of Small,Highly Conserved,Cysteine-rich Proteins

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.The metallothioneins (MTs)¹ are a family of small, highly conserved proteins with the specific capacity to bind metal ions (1–3). Mammalian MTs, typically 61 to 68 amino acid residues in length, contain 20 invariant cysteine residues that form two distinct metal-binding domains. Up to seven or eight metal ions may be coordinated per MT. Many functions have been attributed to this redox-active protein, including zinc homeostasis; heavy metal detoxification; metal exchange; metal transfer; and protection against oxidative damage, inflammatory responses, and other cellular stresses (4–6). Changes in MT expression have been associated with human pathologies including cadmium-induced renal toxicity (7), neurodegeneration (8), and many forms of cancer (9, 10). The understanding of these changes is complicated by the 11 functional MT genes, seven pseudogenes, and four MT-like genes encoded in the genome, most of which contain only small differences in amino acid sequence (11). Seventeen of the 18 genes and pseudogenes are clustered together on chromosome 16, which is known to be enriched for intrachromosomal duplications (12). The various MT gene products differ in their patterns of mRNA and protein expression in human tissues and cell lines. Immunohistochemical detection using antibodies that do not discriminate between MT-1 and MT-2 isoforms indicates wide tissue and cell type distribution of MTs, as illustrated with the MT-1A entry of the Human Protein Atlas (13, 14). Measurements of individual MT mRNA levels, however, clearly demonstrate differential expression of specific MT-1 isoforms in human tissues and cell lines (15–17). The MT-3 (18, 19) and MT-4 (20) mRNAs are expressed in even narrower ranges of cell types.An abundance of immunohistochemical and mRNA measurements show that alteration of MT isoform expression is correlated with a variety of cancers (9, 10). For example, several studies show that the expression of specific MT isoforms is altered in invasive ductal breast carcinomas. Elevated MT-2A (21) or MT-1F (22) is correlated with increased proliferation or tumor grade, respectively. Expression of MT-3 is associated with poor prognosis (23, 24). The MT-1E isoform is found in estrogen-receptor-negative (ER⁻), but not estrogen-receptor-positive (ER⁺), tumors (25) and cell lines (26). Parallel assessment of changes in MT protein expression via immunohistochemistry supports the mRNA data up to a point. Except for antibodies specific for the MT-3 isoform (27), all commercially available MT antibodies are pan-specific for the MT-1, MT-2, and MT-4 protein isoforms (28). This is because epitopes recognized by antibodies raised against MT-1 or MT-2 are limited to the first five residues of the acetylated N terminus, which are invariant among all MT-1, MT-2, and MT-4 isoforms (29–31). This includes the commercially available E9 antibody that has been used to demonstrate the overexpression of MT in a wide variety of human cancers (28, 32, 33). In general, the overexpression of MT in various cancers has been associated with resistance to anticancer therapies and linked to a poor prognosis.The mounting evidence that specific MT isoforms may be useful prognostic and diagnostic markers for cancers highlights the need for alternative approaches to the assessment of MT isoform expression at the protein level. A few mass-spectrometry-based studies have succeeded in identifying the complement of MT isoforms in human cells (34, 35). Though top-down approaches hold promise for the quantitation of MTs based on the unique masses of intact isoforms (34, 36), this has yet to be exploited. Inductively coupled plasma MS has been used to quantify total metal-bound MTs in cells and tissues, but it cannot assign relative abundance values of MT isoforms because the proteins are reduced to their elemental composition with this technique. Thus far, MALDI-MS has been used in parallel with inductively coupled plasma MS for the qualitative identification of isoforms (35). Bottom-up quantitative approaches specifically targeting MTs have not yet been reported.The use of mass spectrometry to quantify MT isoforms is not straightforward. The N-terminal tryptic peptide of each human MT isoform encompasses the only sequence that distinguishes all 12 and therefore may be used for their identification and quantitation in complex biological samples from cells and tissues (34). Any attempt at quantitation of this family of small, highly conserved, cysteine-rich proteins therefore requires reproducible detection of these signature peptides.An optimized bottom-up proteomic method is presented here that is capable of identifying and quantifying all isoforms that constitute the human MT gene family in a single experiment. The approach is comparable in sensitivity and dynamic range to quantitative PCR methods used to measure mRNA levels. Quantitative and qualitative differences between mRNA and protein expression indicate that isoform-specific measurements of protein levels complement and extend our understanding of MT isoform expression in complex biological samples. The method was applied to the characterization of MT isoforms in ER⁺ and ER⁻ breast cancer cell lines. Protein and mRNA measurements showed the same complement of isoform expression, confirming differential MT expression between ER⁺ and ER⁻ cell lines. The mass spectrometry assay further showed dramatic differences in the abundance of protein and mRNA in specific isoforms, an observation that has not been previously reported.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.