Localization of telomerase hTERT protein and hTR in benign mucosa, dysplasia, and squamous cell carcinoma of the cervix.

Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.

Avocado fruit protoplasts: a cellular model system for ripening studies.

Mesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.

Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening.

We present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a lambda EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5' end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5' flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.

Human pepsinogen C (progastricsin). Isolation of cDNA clones, localization to chromosome 6, and sequence homology with pepsinogen A.

The entire pepsinogen C (PGC) coding sequence was determined by analysis of a series of five overlapping cDNA clones identified in a library constructed from human gastric mucosa poly(A+) RNA. A partial cDNA clone was initially identified using a 256-fold degenerate oligonucleotide probe for amino acid residues 4-12 of pepsin C, and subsequently 4 additional clones were identified upon rescreening with a probe complementary to the 5' region of the original cDNA clone. Northern analysis of gastric mucosa poly(A+) RNA with a PGC cDNA probe revealed an mRNA 1.5-kilobase species that was indistinguishable from that detected with a human pepsinogen A (PGA) cDNA probe. In contrast, the PGC and PGA cDNA probes detected distinct genomic restriction fragments indicating there was no detectable cross-hybridization under high stringency conditions. The PGC gene was localized to human chromosome 6 by analysis of a panel of human x mouse somatic cell hybrids. The regions containing the active site aspartyl groups of PGC are conserved in relationship to several other aspartic proteinases. We propose that the absence of detectable immunologic cross-reactivity between the two groups of human pepsinogens, A and C, results from divergent evolution of sequences located on the surface of the zymogens in contrast to the strongly conserved active site regions located within the binding cleft of the enzymes that are inaccessible for antigenic recognition.

Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r.

Mutations in the araC gene have been isolated which alter both the activator and autoregulatory functions of AraC protein (L.G. Cass and G. Wilcox, J. Bacteriol. 166:892-900, 1986). In this study, the effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which was not observed in the araC+ cell. Experiments were therefore focused on understanding the mechanism of this novel activation. We describe a systematic analysis of the effect of mutations within the known regulatory binding sites for araBAD and araC transcription on araC expression. Our results suggest that the novel activation of araC expression requires the AraC activator-binding site, araI, and the cyclic AMP receptor protein-cyclic AMP complex-binding site. We also found that in the absence of L-arabinose, the araI site was required for maximal autoregulation by the wild-type AraC protein.

Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein.

Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation. The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA. The mutant phenotype selected was the inability to autoregulate. The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene. Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein. The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding.

The araIc mutation in Escherichia coli B/r.

The araIc allele is a cis-acting mutation which has been used to define the araBAD promoter in Escherichia coli B/r. Nineteen araIc mutants were originally isolated by Englesberg and co-workers as Ara+ "revertants" of an araC deletion mutant (Englesberg et al. J. Mol. Biol. 43:281-298, 1969). The mutants constitutively expressed araBAD gene products in the absence of functional araC activator protein. Eight of the araIc mutations have been cloned by in vivo recombination onto pBR322-ara hybrid plasmids. Restriction and DNA sequence analysis of these araIc mutations showed that they result from a single base-pair change located at -35 in the araBAD promoter.

DNA sequence of the araC regulatory gene from Escherichia coli B/r.

The DNA sequence of the araC regulatory gene from Escherichia coli B/r has been determined by the base-specific chemical cleavage reactions of Maxam and Gilbert. An open reading frame is found which codes for a protein of 292 amino acids. A nonsense mutation, araC5, is shown to result from a G to A transition at nucleotide 429 converting the tryptophan codon TGG to the amber codon TAG. A deletion which does not recombine with any known point mutation in araC, delta(araCO)719, removes all but the last 22 codons of the gene.

The araC regulatory gene mRNA contains a leader sequence.

An estimation of the size of the araC gene in Escherichila coli B/r was made by sub-cloning restriction fragments of the araC-containing hybrid plasmid pTB1 into the plasmid pBR322. Plasmids which contained a functional araC gene were identified by genetic complementation tests. DNA sequence analysis of the promoter-proximal region of the araC gene revealed that araC mRNA contains a 150 nucleotide leader.