A proteomic approach for the discovery of early detection markers of hepatocellular carcinoma.

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to "cherry pick" or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Sequence and developmental regulation of the gene that encodes the Dictyostelium discoideum L3 ribosomal protein.

We have isolated and characterized genomic and cDNA recombinant plasmids that encode the Dictyostelium discoideum (Dd) ribosomal protein L3 (rpL3). Genomic plasmids were identified using a probe derived from the Saccharomyces cerevisiae TCM1 gene, that encodes the yeast rpL3. The DdL3 gene contains two introns and encodes a protein 398 amino acids in length that shows a high degree of homology to the conserved rpL3 protein of both lower and higher eukaryotes. During development, both the pattern of accumulation of DdL3 mRNA and changes in its translational activity are identical to those observed for other r-protein mRNAs.

Comparative analysis of the sequence and structure of two Drosophila melanogaster genes encoding vitelline membrane proteins.

Two Drosophila melanogaster vitelline membrane protein-encoding genes (VM), located at polytene band positions 26A and 34C, have been cloned and comparatively characterized at the nucleotide level. Sequence analysis of genomic and cDNA clones for the two genes, VM26A.1 and VM34C.1, indicates that both are similarly organized with a central highly conserved domain [Scherer et al., Dev. Biol. 130 (1988) 786-788] which is flanked by unrelated regions, and that both genes lack introns. Comparison of the upstream regions reveals that both VM genes contain a hepatmeric element identical to one associated with the D. melanogaster yolk protein-encoding genes (YP). This heptamer occurs in the specific 5' flanking region responsible for ovarian temporal- and tissue-specific control in both VM and YP genes. A putative chorion transcription factor 2 site is also associated with an upstream control element of VM26A.1, but not with any sequenced portion of VM34C.1.

Sequence elements that affect mRNA translational activity in developing Dictyostelium cells.

Post-transcriptional controls, including changes in both mRNA translational activity and stability, play an important role in the regulation of ribosomal protein gene expression in developing Dictyostelium discoideum cells. Previously we have shown that the mechanisms which regulate the translational activity of the r-protein mRNAs operate at the level of translational initiation and do not involve changes in polyadenylation or capping. By analysing the translational behavior of chimeric and mutant mRNAs in transformed cells, we have now been able to localize the determinants of translational activity of one of the r-protein mRNAs to the 5'-untranslated region. Although this and other r-protein mRNAs differ strikingly from the Dictyostelium consensus in the region of the initiator AUG codon, we find that improving the match to that consensus does not increase the translational activity of the message in developing cells. Current experiments are designed to determine whether translational regulation is mediated by strong interactions with specific inhibitors or by weak interactions with translational initiation factors.

Regulation of mRNA stability and the poly(A) problem in Dictyostelium discoideum.

This paper reviews our studies of three aspects of post-transcriptional regulation in Dictyostelium discoideum: 1) the determinants of mRNA stability in vegetative amoebae; 2) the effects of disaggregation and cyclic AMP on the decay rates of cell-type-specific mRNAs in late developing cells; and 3) the cytoplasmic function of the 3' poly(A) tracts present on most mRNAs. We find that: 1) mRNA stability in vegetative amoebae is not dependent on mRNA size, ribosome loading, or poly(A) tract length, but may be determined by specific 3'-untranslated sequences within a given mRNA; 2) mRNA decay rates in late developing cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs; and 3) poly(A) is most likely involved in the initiation of protein synthesis via an interaction with cytoplasmic poly(A)-binding proteins.

Post-transcriptional regulation of ribosomal protein gene expression during development in Dictyostelium discoideum.

We have isolated recombinant plasmids that contain cDNA inserts complementary to mRNAs encoding six different r-proteins of Dictyostelium discoideum. Southern and quantitative dot blot analyses have shown that each of the r-protein genes represented in these plasmids is encoded by a single copy gene and that these genes are not tightly linked to each other. We have determined the relative amount of the six r-protein mRNAs present in cells at intervals throughout development and find that for the first 9 hours of development, each of the mRNAs remains present at virtually the same level as in vegetatively growing cells. Between 9 and 11 hours of development, there is a rapid loss of these mRNAs to 15% or less of vegetative levels, and that low level remains, or slightly declines, through the late stages of development. We have shown that two post-transcriptional events contribute to the developmental regulation of the expression of the r-protein genes. The first involves a specific block to translational initiation that is not the result of inactivation of these mRNAs by decapping or deadenylation. The second is a change in the stability of these mRNAs during early development. In order to begin to analyze the role of specific sequences that may act as targets or signals in these events, we have cloned and sequenced a 1.9-kb genomic DNA fragment that encodes one of the r-proteins. We find that transcription of this gene begins in a pyrimidine-rich region that is not preceded by a TATA box, the gene contains a single intron of 350 bp, and there are two alternative 3' processing sites. In addition, the 5'-untranslated region of the transcript contains an unusually high percentage of G and C residues relative to other Dictyostelium mRNAs.

Nucleotide sequence and characterization of the transcript of a Dictyostelium ribosomal protein gene.

Dictyostelium ribosomal protein mRNAs are subject to developmental regulation of both their translation and their stability. In order to consider whether such post-transcriptional regulation can be attributed to structural features of the mRNAs, we have cloned and sequenced a 1.9 kb EcoRI genomic DNA fragment which contains the gene for the Dictyostelium ribosomal protein 1024 (rp1024). The rp1024 gene contains a single intron of 350 bp which begins just after the fourth codon of protein coding sequence. Transcription begins 11 to 28 bp upstream from the initiator ATG in a pyrimidine rich region which is preceded by an oligo(dT)10 stretch, but which lacks a TATA box in the expected position. Processing of the 3' end occurs at either of two sites, resulting in two types of transcript which are present in equimolar amounts in both vegetatively growing and developing cells. Therefore, their relative abundance shows no correlation with the changes in translatability and stability of r-protein mRNAs which occur during development. A comparison of the sequence of the 5'-untranslated region of rp1024 mRNA to those of other Dictyostelium mRNAs shows that it differs significantly, primarily in its relatively high G+C content.

Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development.

Throughout the developmental program of Dictyostelium discoideum there are substantial changes in the rates of both ribosome utilization and rRNA transcription and processing. We examined the regulation of ribosomal protein (r-protein) gene expression and found that, at the start of development, expression of these genes was drastically and specifically reduced by a block to translational initiation. An apparently separate event signals a sudden decrease in the relative amount of r-protein mRNA at about 10 h of development, a time when aggregated amoebae are forming tight cell-cell contacts. For the first 9 h of development, the relative amount of r-protein mRNA remained essentially unchanged and comparable to levels detected in growing cells. While the r-protein mRNAs were almost fully loaded on polysomes during vegetative growth, they were specifically excluded from polysomes at the start of development. The translational block was not the result of irreversible structural changes which inactivate the r-protein mRNAs since they remained translatable both in vitro, in wheat germ extracts, and in vivo, where they were recruited onto polysomes in the presence of the elongation inhibitor cycloheximide. In addition, precise measurements of poly(A) tail lengths on individual hybrid-selected mRNA species showed that there is no difference in the poly(A) tail length of r-protein mRNA isolated from growing cells and 1-h developing cells. Therefore, changes in translational efficiency cannot be attributed to cleavage of poly(A) tails.

Ribosomal proteins are encoded by single copy genes in Dictyostelium discoideum.

Five recombinant plasmids which encode ribosomal proteins (r-proteins) from Dictyostelium discoideum have been isolated. Poly(A) + RNA was size-fractionated by preparative agarose gel electrophoresis and a fraction encoding proteins of less than 35 kDa was used to construct a cDNA library in the plasmid vector pBR322. Individual clones from the library were screened by hybrid-selected translation and those encoding r-proteins were identified by co-migration of the translation products in two-dimensional gel electrophoresis with marker proteins purified from Dictyostelium ribosomes. Initial characterization using the five cDNA plasmids indicates that these r-proteins are encoded by single copy genes and that they are not tightly clustered in the genome.

Intron bypass: a rapid procedure for eliminating introns from cloned genomic DNA and its application to a cellulase gene.

We have devised a DNA cloning procedure in which the introns present in a genomic DNA fragment can be eliminated easily and rapidly. The technique combines the methods of cDNA and genomic cloning in a way which assures full-length representation of the intron-free transcript. Moreover, plasmids made by this technique can be designed to contain flanking untranscribed regions which may play a role in the regulation of expression. One strand of a linearized plasmid containing the 3'-end of a gene is used to prime cDNA synthesis from an annealed mRNA template. A second plasmid containing the 5'-end of the gene is linearized, denatured, and annealed to the extended 3'-end molecules and the resulting circular, partial duplexes are used to transform bacterial cells. Two different recombinant plasmids which contain DNA encoding the cellulase, exocellobiohydrolase I, from Trichoderma reesei have been constructed using this method. They both contain the entire translated region of the gene uninterrupted by introns. One plasmid contains additional DNA at the 5'-end, including approximately 150 bp 5' to the start of transcription. The inserts of both plasmids can be excised in one piece.

Lung cancer. Improved cytologic detection by inducing production of sputum.

The principle of producing bronchial lavage by deposition of large amounts of heated aerosol has resulted in a significantly greater yield of positive cytologic diagnosis of bronchogenic carcinoma than with repeated "volunteer" specimens of sputum. Positive pressure plus bronchodilators augments greater sputum volume. Using this technique, cases in which results of bronchoscopy and aspiration biopsy were negative for malignant change, were diagnosed cytologically. Application of this technique can sometimes detect early lung carcinoma before roentgenographic changes are detectable. Positive tests in clinically far advanced disease may prevent unnecessary surgical intervention. The simplicity of the technique, the freedom from adverse reactions, and its wide acceptance by the subjects tested, make it valuable in the diagnosis of lung cancer.