Choice of labeled precursors in synthesis of DNA for molecular hybridization.
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Animals↗
Biomedical subjects
D R Roop
Publications and source records attributed to D R Roop.
Transcription of structural and intervening sequences in the ovalbumin gene and identification of potential ovalbumin mRNA precursors.
Structural sequences that are extensively separated by nonstructural intervening sequences in the natural ovalbumin gene are coordinately expressed in target and nontarget tissue. The intervening sequences, which consist of unique sequences in the chick genome, are transcribed in their entirety. The amount of nuclear RNA corresponding to these sequences, however, is approximately 10 times less than that observed for structural sequences. The accumulation of RNA corresponding to structural and intervening sequences during acute estrogen stimulation suggests either that there are different rates of transcription for these regions of the ovalbumin gene or that RNA sequences corresponding to the intervening sequences are preferentially processed and degraded. Comparison of the in vitro expression of portions of the ovalbumin gene in nuclei isolated from chronically stimulated oviducts indicates that both structural and intervening sequences are preferentially transcribed in vitro at rates approximately 500 times greater than expected for random transcription of the haploid chick genome. In addition, electrophoresis of oviduct nuclear RNA on agarose gels containing methylmercury hydroxide reveals multiple species of RNA that are from 1.3 to over 4 times larger than ovalbumin mRNA and hybridize to both structural and intervening sequences of the ovalbumin gene. These results are consistent with transcription of the entire ovalbumin gene into a large precursor molecule followed by excision of the intervening sequences and appropriate ligation of the structural sequences to form the mature mRNA.
Animals↗
Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes.
We have used immunoelectron microscopy to map the biosynthetic pathways of loricrin and filaggrin in epidermal keratinocytes at successive stages of differentiation in newborn mouse skin. The filaggrin epitope is first detected in large, irregularly shaped, keratohyalin granules (F-granules) in the stratum granulosum, and then distributed throughout the cytoplasms of the innermost layers of stratum corneum cells. We conclude that the poly-protein filaggrin precursor is first accumulated in F-granules, from which it is subsequently released and processed into filaggrin, and becomes associated with the densely packed bundles of keratin filaments inside stratum corneum cells. Its diminished visibility in the outer layers correlates with the known degradation of filaggrin to free amino acids. Loricrin is first detected in small round keratohyalin granules (L-granules), and subsequently at the periphery of cells throughout the stratum corneum. Labeling of purified keratinocyte envelopes establishes that this loricrin epitope is exposed only at their inner (cytoplasmic) surface. Thus loricrin is initially accumulated in L-granules, to be released at a specifically programmed stage of keratinocyte maturation, and incorporated into the covalently cross-linked lining of the cell envelope. Since loricrin is rich in cysteine, L-granules account for the sulfur-rich keratohyalin granules described earlier. Proposals are made to rationalize why, subsequent to synthesis, filaggrin precursor and loricrin should be segregated both from each other and from the rest of the cytoplasm.
Amino Acid Sequence↗
An activated Harvey ras oncogene produces benign tumours on mouse epidermal tissue.
Studies of the mutagenic action required for specific chemicals to produce benign or malignant tumours suggest that in mouse skin at least two genetic events occur before carcinoma formation. The isolation of an activated form of the c-rasH gene from skin papillomas has provided evidence that this gene may be a target for the first mutation, which could constitute the initiating mutation in skin carcinogenesis. In vitro studies indicate that the v-rasH gene of Harvey murine sarcoma virus (Ha-MSV), a replication-defective transforming retrovirus, could impart a conditional initiated phenotype on cultured keratinocytes by blocking their ability to differentiate terminally and arresting them in a late basal cell stage of maturation. We now show that when the Ha-MSV v-rasH gene is introduced into cultured keratinocytes by a defective retroviral vector, skin grafts constructed with cells carrying the mutated ras oncogene produce papillomas on athymic nude mouse recipients. Furthermore, the expression of the exogenous oncogene seems to be regulated at the transcriptional level in the differentiated portions of the benign tumour.
Animals↗
Transduction of a preselected population of human epidermal stem cells: consequences for gene therapy.
Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.
Adult↗