Expression of the hepatocyte growth factor-like protein gene in human hepatocellular carcinoma and interleukin-6-induced increased expression in hepatoma cells.

Human hepatocellular carcinoma is one of the most frequent malignant tumors. It may occur following exposure to various agents, including viruses and chemical carcinogens; however, the underlying mechanisms of the hepatocarcinogenesis are not known. The present study is the result of our search for genes which may be abundantly expressed in human primary liver carcinoma. One of these genes was found to encode the human hepatocyte growth factor-like protein (HGFLP), also known as macrophage-stimulating protein. HGFLP is structurally homologous to hepatocyte growth factor, a potent growth factor for liver. HGFLP mRNA was also found to be overexpressed in a hepatoblastoma sample and in a sample of subacute fulminant hepatic necrosis. In a study on the effects of cytokines on the expression of HGFLP, we found that IL-6 increased expression of HGFLP mRNA in Hep G2 cells, but IL-1alpha, IL-1beta and TNF-alpha had no effect. An increase in HGFLP could be the result of inflammation and/or tissue injury and its overexpression may prove to be useful as an indicator of hepatoma.

Regulation of human Gc (vitamin D--binding) protein levels: hormonal and cytokine control of gene expression in vitro.

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group-specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGF beta) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGF beta suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

The vitamin D-binding protein gene: quantitation of amplified nucleic acids by ELISA.

We have utilized enzyme-linked immunosorbent assay (ELISA) to quantitate PCR-amplified DNA. This method was used to measure mRNA for the vitamin D-binding protein (Gc), beta-actin and the transferrin receptor (TR) gene in the Hep3B cell line. Total RNA from Hep3B cells was reverse transcribed to obtain cDNA, which was amplified in the presence of digoxigenin-dUTP by PCR. The PCR products were then hybridized in liquid phase to a biotinylated, nested capture probe for the respective sequences. The hybridized products were bound to a streptavidin-coated ELISA plate and were detected by an alkaline-phosphatase-conjugated antibody to digoxigenin. ELISA standard curves for Gc and control genes, beta-actin and TR, were obtained after PCR amplification of serial dilutions of Hep3B total RNA. As an external standard, an ELISA standard curve for Gc was obtained after PCR amplification of serial dilutions of a full-length Gc cDNA insert obtained from a recombinant plasmid. Thus, we were able to develop a non-isotopic quantitation assay for PCR-amplified DNA that is highly sensitive and has the specificity of hybridization-based methods.

Construction of mRNA genes for the synthesis and translation of duck alpha globin mRNA.

In studies of the effects of changes in mRNA structure and sequence on the initiation of protein synthesis, we used a generally applicable approach to transcribe reconstructed genes for duck alpha A globin by the bacteriophage SP6 RNA polymerase promoter in a pGEM-2 plasmid vector. The genes were reconstructed such that the first nucleotide to be transcribed, the 5' adenosine, was placed directly adjacent to the SP6 promoter sequence. The 3' ends of the genes were constructed such that cleavage with Ssp 1 endonuclease yielded a template that directed the synthesis of mRNA terminating in a poly A tail containing 56 adenosines and a single 3' uridine. Special conditions using a Mn++ buffer were developed to enable the SP6 RNA polymerase to initiate at the 5' adenosine and synthesize the A-start transcription product. The mRNA could be capped and was subsequently used as an effective template for in vitro translation and synthesis of duck alpha A globin.

Displacement synthesis of globin complementary DNA: evidence for sequence amplification.

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.

Globin proteins of the normal and anemic duck.

The red blood cells of normal adult ducks contain two main hemoglobins. The most abundant type, HbA, comprises approximately 80% of the total, with the remaining 20% being made up of HbD. An attempt was made to determine whether during hemolytic anemia a special alpha globin chain (alpha s) replaces the alpha chain of HbA found in normal animals. This special stress alpha globin, whose existence has been seriously questioned, was originally postulated to explain the sequence discrepancies obtained between alpha chains of normal and anemic chickens and ducks. Using gel electrophoresis, isoelectric focusing, and HPLC peptide mapping techniques no qualitative differences between the alpha A globins of normal and anemic animals were found. The nature of the beta globin chains present in adult ducks has also never been rigorously established. In this work, a variety of techniques, including HPLC, gel electrophoresis, and microcolumn amino acid analysis, were used to examine the beta chains from each hemoglobin. Using these methods, no differences were found between the beta globin chains of the two hemoglobins.

Characterization of duck genome fragments containing beta and epsilon globin genes.

We have isolated two allelic duck-genomic DNA fragments containing beta-type globin genes. The beta- and epsilon-globin genes are 1800 bp apart in both fragments, have second introns of 1100 and 1200 bp, respectively, and small first introns. We have also determined approx. 550 nucleotides of the sequence on the 5' side of the beta-globin gene. Comparison with the chicken beta-globin gene suggests sequences of possible significance to gene expression.

Nucleotide sequences for the duck globin mRNAs.

Normal adult ducks possess two main types of hemoglobin: a major species (80%), HbI (alpha I2 beta I2), and a minor species (20%), HbII (alpha II2 beta II2). We have cloned recombinant cDNAs for the duck globin mRNAs using the ribosubstitution floppy loop technique. We present here the sequence for a duck alpha globin mRNA closely related to the chicken alpha D globin mRNA. Our new sequence data also include the 5' noncoding regions of the duck alpha A, alpha D, and beta globin mRNAs. Analysis of the untranslated regions of these mRNAs reveals several conserved sequences which may be important in the regulation of gene expression.

Dialyzable leukocyte extracts contain thymosin.

Trypsinization of human T-lymphocytes removes surface receptors which bind to sheep erythrocytes (E). Human dialyzable leukocytes extracts (DLE) and thymosin (Fraction V) have been shown to significantly increase the rate of regeneration of T-lymphocyte E-receptors. Both physical-chemical and immunochemical results reported herein indicate that the enhancing effect of human DLE preparations on the rate of regeneration of T-lymphocyte E-receptors is due at least in part to the presence of thymosin alpha 1-peptide in these preparations. Thymosin alpha 1-peptide purified from thymosin Fraction V and putative thymosin alpha 1 preparations purified from human DLEs were each active not only in increasing the rate of regeneration of T-lymphocyte E-receptors removed by trypsinization but also were active in vitro in markedly increasing the number of E-rosetting cells in two patients with immunodeficiency disease manifested in part as a reduction in the normal percentage of mature T-lymphocytes capable of forming E-rosettes.

Bovine 'transfer factor': an oligoribonucleopeptide which initiates antigen-specific lymphocytes responsiveness.

Bovine transfer factor (TF)--active in initiating specific responsiveness in human thymus-derived (T) lymphocytes to purified protein derivative from Mycobacterium tuberculosis (PPD) in vitro--was partially purified from the dialyzable portion of medium from immune lymph node cells (DLNE). Its physiochemical properties and structure were determined by methods previously employed to characterize human PPD-specific TF isolated from dialyzable leukocyte extracts (DLE). Bovine TF had a molecular weight (MW) of 1100-3000, was destroyed by heating at 56 or 80 degrees C for 30 min, was soluble in water but not in phenol or ether, and could be precipitated with ethanol. Bovine TF activity eluted as a single peak after high-pressure reverse-phase liquid chromatography (HPLC); the active moiety contained at least one free co-planar cis-diol group, as shown by boronate affinity chromatography. Additional structural features were deduced by evaluating TF activity after incubation with various endonucleases, exonucleases, and peptidases, a phosphatase, and a protease. The combined results indicate that bovine TF specific for PPD is an oligoribonucleopeptide. A simplest case molecular model was constructed on the basis of the data obtained. A comparative evaluation of the physicochemical properties and structural features of bovine TF and human TF specific for PPD indicated striking similarities and some differences.

Nucleotide sequence for a novel duck alpha-globin gene.

Nucleotide sequence analysis of a recombinant cDNA for a duck alpha-globin gene indicates a gene with novel features and evolutionary history. The duck alpha-globin double-stranded cDNA, treated with S1 nuclease and tailed with poly(dC), was inserted into the PstI restriction endonuclease site of pBR322 tailed with poly(dG). Nucleotide sequence analysis of the resulting recombinant cDNA indicates that it contains the translated region and all of the 3' untranslated region for an alpha-globin gene. The sequence was determined by procedures designed especially for rapid analysis of pBR322 (PstI site) recombinant cDNAs. This duck globin has sequences related to the chicken alpha-A globin in some regions but in other regions it is more closely related to another alpha globin found in anemic chickens.

Effects of dialyzable leukocyte extracts with transfer factor activity on leukocyte migration in vitro. V. Antigen-specific lymphocyte responsiveness can be initiated by two structurally distinct polyribonucleopeptides.

Human transfer factors (TF) active in specifically inducing responsiveness in human thymus-derived (T) lymphocytes previously nonresponsive to purified protein derivative from Myobacterium tuberculosis (PPD) or to Coccidioides immites (Cocci) in vitro were isolated from the dialyzable portion of extracts of immune leukocytes (DLE). Each TF segregated into two active fractions after high-pressure reverse-phase liquid chromatography (HPLC), suggesting the presence of two TF components in DLE for each antigen specificity. Determination of the structures of both TF components specific for PPD was accomplished by evaluating their activity after incubation with various endonucleases, exonucleases, phosphatases, peptidases and a protease. The results indicated that both PPD-specific TF components are oligoribonucleopeptides but that they are structurally distinct. Simplest-case molecular models were constructed on the basis of the data obtained.

The primary sequence of rabbit alpha-globin mRNA.

The rabbit alpha-globin DNA insertion in the chimeric plasmid pHb 72 (Liu et al., 1977) has been sequenced by the method of Maxam and Gilbert (1977). This has enabled us to determine the messenger RNA(mRNA) sequence beginning in the 5' untranslated region 9 nucleotides before the initiation codon and extending through the first 361 nucleotides of the translated region. The data reported here overlap and are in complete agreement with sequences determined by Baralle (1977) for the 5' end of the mRNA and by Proudfoot et al. (1977) for the 3' end. Our sequence is also in agreement with the partial complementary RNA (cRNA) sequencing data which we reported previously (Paddock et al., 1977), this work marks the completion of the primary sequence of the rabbit alpha-globin mRNA. These observations reaffirm the high fidelity with which gene copies can be synthesized in vitro, cloned in a bacterial plasmid and maintained in the host. The general features of the mRNA nucleotide sequence are duscussed with particular attention given to the base composition and codon preferences observed and to comparison of this sequence with other completed mRNA gene sequences. A new computer program has been used to search for the most stable base-pairing arrangement of the completed mRNA.