Specific and sensitive mRNA biomarkers for the identification of skin in 'touch DNA' evidence.

In forensic casework analysis it is often necessary to attempt to obtain DNA profiles from microscopic amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is routinely demonstrated with so-called 'touch DNA' evidence, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. Although a genetic profile from trace biological evidence is routinely obtained, the tissue source of the profile is rarely known. This merely perpetuates the 'mystery' of the nature of 'touch DNA' evidence allowing the significance or meaningfulness of genetic profiles obtained from these samples to be challenged. Numerous reports state that the tissue source of origin of 'touch DNA' evidence cannot be determined due to the small amount of biological material present, while others conclude that the DNA profiles are obtained from shed skin cells (as opposed to, say, buccal epithelial cells present in saliva traces) without any scientific basis for this assertion. Proper identification of the biological material present might be crucial to the investigation and prosecution of a criminal offense and a misrepresentation of the nature of the evidence can have undue influence on the perception of the circumstance of the crime. Thus far, research has failed to provide forensic scientists with feasible, definitive methods to identify the tissue origin of 'touch DNA'. In the present work, we sought to identify novel highly specific and sensitive messenger RNA (mRNA) biomarkers for the identification of skin. Gene candidates were identified using both literature searches and whole transcriptome deep sequencing (RNA-Seq). Utilizing this dual approach, we identified and evaluated over 100 gene candidates. Five mRNA markers were identified that demonstrated a high degree of specificity for skin. Using these markers, we have been able to successfully detect and identify skin using as little as 5-25 pg of input total RNA from skin and, significantly, in swabs of human skin and various touched objects. One of the markers, LCE1C, is particularly highly sensitive and was detected in the majority of skin samples tested including touched objects. We have been successful in incorporating the five skin biomarkers into two multiplex systems. Although further work is needed to optimize the assay for routine casework, the initial studies demonstrate that a molecular-based characterization of the biological material recovered from touch samples is possible.

Making decisions through Myc.

c-Myc is a transcriptional regulator involved in carcinogenesis through its role in growth control and cell cycle progression. Here we attempt to relate its role in stimulating the G1-S transition to the ability to affect functioning of key cell cycle regulators, and we focus on how its property of modulating transcription of a wide range of target genes could explain its capacity to affect multiple pathways leading to proliferation, apoptosis, growth, and transformation.

Design and properties of a Myc derivative that efficiently homodimerizes.

bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

Ras oncogene transformation of human B lymphoblasts is associated with lymphocyte activation and with a block of differentiation.

The p21ras small GTP binding proteins participate in signal transduction from cell surface receptors and affect neoplastic transformation and development in many different cell types. In the present study, we examined the relationship between ras transformation and differentiation of human B lymphocytes. We show that the constitutive expression of the T24 Ha-ras oncogene in EBV-immortalized B lymphoblasts was associated with the induction of the interleukin 2 receptor alpha subunit, with an impaired immunoglobulin gene expression, altered adhesion properties and increased survival in serum-free medium. Since induction of the IL-2 receptor alpha subunit is a hallmark of lymphocyte activation, we suggest that p21ras naturally triggers B cell activation. The ras-transformed lymphocytes displayed a fully functional IL-2r, as assessed by c-fos induction following treatment with IL-2; nevertheless, they were not growth stimulated by this lymphokine. The decreased expression of immunoglobulin genes indicates that the ras oncogene blocks terminal differentiation to plasma cells, possibly by inhibiting the activity of lymphocyte-specific transcription factors. Somewhat unexpectedly, the constitutive p21ras activity did not cause an increased DNA binding of transcription factors PEA1 (AP1), PEA3, Oct-2 or NF-kB.

Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene.

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.

Studies on host-virus genome relationship in Epstein-Barr virus immortalized lymphoblastoid cell lines.

Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.

Fate of Friend leukaemia cells and lymphoma cells injected into mouse blastocysts.

Our aim was to verify whether differentiated murine cells grafted into mouse blastocysts would follow the same fate as teratocarcinoma cells which lose their malignant character and participate in the development of the host embryo yielding chimaeric animals. Two combinations of host embryo-cancer cells were used. Animals were examined after birth or by pregnancy interruption at various stages of development. More than 350 animals obtained from cancer-cells-injected embryos failed to show direct evidence of loss of malignancy.

Study on the participation of neoplastic cells in the development of mouse embryos.

To test the ability of cloned committed erythroleukemic cells to participate in development we have injected. Friend leukemia cells (FLC) into C57Bl/6 mouse blastocysts together with Friend leukemia virus (FLV) and we have examined the newborn individuals derived from them. Five animals out of 32 born have FLC-derived neoplasia. The incidence of neoplasia is increased as compared with other similar experiments without the virus. In two of the animals with the FLC neoplasia the disease manifestation is an erythroid leukemia similar to the one obtained directly with the virus in normal DBA/2 mice.

Mechanism of phage Mu-1 integration: nalidixic acid treatment causes clustering of Mu-1-induced mutations near replication origin.

Frequencies of Mu-1-induced mutants of Escherichia coli have been compared under two different experimental conditions: cells in exponential growth and the same cells treated with nalidixic acid. The average of values obtained from the nalidixic acid-treated culture is 3 times higher than that obtained from the control. Individual ratios of the frequency of mutants in the two cultures yield decreasing values from 6 to 1, starting from the point of origin of DNA replication to the termini of DNA replication. These results are compatible with the idea that Mu-1 integrates at the replication fork.