Search

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.

Publisher Summary Colony hybridization has been carried out using several types of supports. Nylon membranes are the most durable with regard to resistance to damage and repetitive use but are also the most expensive and require many steps similar to those required for nitrocellulose. Nylon membranes from different manufacturers require different treatments and the instructions of the manufacturer must be followed closely for optimal results. In comparison to other hybridization supports, several steps—such as baking in a vacuum oven, prehybridization, and some probe purification steps—can be eliminated by using the labeling procedure described in the chapter. Signals for a perfect match are about l0 to 20 times stronger than those for a singlebase internal mismatch.

A displacement synthesis procedure was used to construct symmetrical recombinant cDNAs. A double-stranded cDNA containing a hairpin loop was extended by the addition of a homopolymer to the 3′ end. This was followed by displacement, or “third strand” synthesis that was primed by an oligonucleotide hybridized to the homopolymer. Ideally, the product should be a DNA containing an inverted repeat with twofold rotational symmetry about nonsymmetrical sequences representing the hairpin loop in the original double-stranded cDNA. Duck globin cDNAs were synthesized by the displacement mode of construction and cloned in pBR322. An α-globin recombinant, pDGPα-2, was isolated and sequenced. This recombinant was found to have two 3′ half regions (mRNA sequence sense) inverted about a nonsymmetrical 5′ half region and an adjacent oligo(dGṡdC) homopolymer. The sequence arrangement indicates that the cDNA folded back on itself, forming a large loop, to prime synthesis of a second strand. We propose that the internal oligo(dGṡ dC) arose through dynamic shifts in cDNA intrastrand structure during the course of synthesis.

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 32 P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S 1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.

Rabbit alpha globin gene copies have been made, using reverse transcriptase and DNA polymerase I, and cloned in bacterial plasmids. Plasmid pHb72 has been shown to contain the alpha gene sequence by restriction enzyme analysis and nucleotide sequencing studies, and therefore has been approved for propagation under P2 plus EK1 conditions by the National REcombinant DNA Committee.

A 169-nucleotide region from the rabbit beta globin gene has been sequenced by analysis of complementary DNA's cloned in bacterial plasmids. Comparison of these sequences with those established for this gene by other techniques provides evidence of a high degree of fidelity and allows the unambiguous establishment that these plasmids do not contain harmful sequences.

Publisher Summary This chapter presents immunochemical and physical–chemical evidence for the presence of thymosin alpha1-peptide in dialyzable leukocyte extracts (DLE). In a study described in the chapter, the collective results obtained from immunologic and physical–chemical evaluations of the E-receptor regeneration promoting activity present in human DLE indicate that it is partly because of the presence of thymosin α1 peptide in DLE. There are, however, additional components in DLE that express a similar activity but do not seen to be intact thymosin α1. Included here might be thymic humoral factor, which has a pi of 5.7 and could be responsible for the activity found in the β region when DLE Sephadex G-25 Fraction IV was fractionated by IEP in a 2.5 to 10 gradient.The documentation of the presence of a variety of T-lymphocyte maturation or differentiation factors in DLE explains why DLE may have nonspecific beneficial effects when it is employed as an immunotherapeutic agent.

Complementary DNA transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded has been inserted into E. coli plasmid pSC 101 by the poly-dT/poly-dA “tailing” and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. Some measure of the amount and identity of inserted globin sequences has been determined by fingerprinting fragments of 32p-labeled globin RNA hybridized to the purified plasmid chimeras.

Nucleotide sequence data for the duck alpha D globin gene indicate an extremely short 3′ untranslated region of only 49 nucleotides. Evolutionarily conserved sequences defined short regions of potential functional significance. Comparisons among sequences for duck and chicken alpha globins indicate that this region has importance in the regulation of globin gene expression.

Publisher Summary This chapter discusses the form(s) of transfer factor (TF) that are released by viable leukocytes from tuberculin responsive human donors when their leukocytes are incubated with PPD in vitro . Data obtained from studying this antigen-liberated (or released) has provided further insight into the roles of TF–H5, TF–H7, and dephosphorylated TF–H5 in the induction of antigen specific responsiveness. The chapter concludes that when immune lymphocytes are incubated with specific antigen they release only dephosphorylated TF–H5. Dephosphorylated TP–H5 is apparently derived from the cell surface (or secreted after it is derived from TF–H5) and also is apparently released nonspecifically by lymphocytes incubated for prolonged periods at 37°C as part of a normal turnover process of cellular membrane constituents. It is speculated, however, that cell-free supernatants derived from lymphocytes merely incubated at 37°C for 4 h without specific antigen added would contain a family of TFs representing the range of antigen sensitivities found in the donor.

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32 P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T 1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the α or β globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants.

We have examined alternative approaches to existing methods for synthesizing complementary DNA suitable for molecular cloning. One model of construction is presented in which ribonucleotides are added to the 3′ end of complementary DNA prior to synthesis of the second DNA strand. The hairpin structure at one end of the molecule is then opened by treatment with RNaase or alkali. This method would eliminate the normal requirement for single-strand specific nucleases and thus shows promise as a means for preserving the 5′ end sequences of mRNA in recombinant complementary DNA studies. Another technique for constructing complementary DNA is proposed in which no cleavage step is required. Instead, a hairpin, double-stranded DNA is extended with a homopolymer at the 3′ end, and displacement or “third-strand” synthesis by the Klenow fragment of DNA polymerase I is primed by an oligonucleotide hybridized to the homopolymer. The end result should be an inverted repeat with two-fold rotational symmetry. The mRNA 5′ end sequences represent the center of symmetry. Cloned, symmetrical DNA should facilitate subsequent nucleotide sequence analysis. Furthermore, the symmetrical molecule may serve as an intermediate in continued DNA synthesis provided the homopolymer chain is sufficiently longer than the primer, thus leading to mRNA sequence amplification in vitro . Alternative options with attendant advantages and disadvantages are given at each stage in the construction schemes. These strategies, along with established procedures, offer a repertoire from which researchers may select in order to fill their specific needs.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

Publisher Summary The dialyzable leukocyte extracts (DLE) prepared from burros immunized with either Mycobacterium tuberculosis (PPD–S) or Coccidioides immitis (spherulin) antigen can transfer antigen-specific cell-mediated immunity (CMI) to previously nonresponsive human lymphocytes in vitro. The results of experiments described in this chapter indicate that when Burros are immunized with either Mycobacterium tuberculosis antigen or Coccidioides immitis antigen, DLE subsequently prepared from their PBL contains TF activity, which is active in inducing antigen-specific responsiveness in previously nonimmune human lymphocytes. Burro TF activity easily can be detected using the LMI assay, the in vitro assay for human TF activity. Characterization of Burro TP specific for PPD–S indicates that it has physical–chemical properties similar to those of human TF derived from DLE.

Deoxynucleotides have been incorporated into RNA synthesized in vitro by RNA polymerase with either double-stranded or single-stranded DNA as a template. By use of this technique to block or promote cleavage at a particular phosphodiester bond, a variety of specific cleavages may be obtained with the available ribonucleases and deoxyribonuclease I. These methods should greatly increase the ease and rapidity of nucleotide sequence determinations.

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.

Nucleotide sequence analysis and restriction endonuclease mapping have been used to characterize a cDNA copy of immunoglobulin MOPC 21 Kappa mRNA clones in the bacterial plasmid pMB9. Three regions of the inserted cDNA of plasmid pL21-1 have been sequenced and match the known protein sequence at amino acid residues 1-24, 128-138 and 171-179. With these sequences to provide absolute correlations between the restriction map and the structural gene sequence it has been possible to exactly deduce the positions of all 11 of the insert restriction sites mapped within the structural gene. The pL21-1 insert contains the complete variable and constant regions as well as parts of the 3' untranslated and polypeptide leader coding sequences.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.

Publisher Summary This chapter presents data on the structures of both human transfer factor (TF) moieties derived from dialysates of leukocyte extracts (DLE) and on the radioactive labeling and subsequent further purification of human TF–H7. It describes experiments in which human TF–H5 and TF–H7 are purified from crude DLE by Sephadex G-25 chromatography, phenol extraction, ethanol precipitation, HPLC, cellulose TLC, and boronate affinity chromatography. In some cases, cellulose TLC was substituted for high-pressure reverse phase liquid chromatography and phenol extraction followed by ethanol precipitation was substituted for Sephadex G-25 chromatography. Bovine TF–C5 was similarly purified from material released by immune lymph node cells. Poiyethyleneimine cellulose (PEI) TLC was performed using ascending homochromatography at room temperature without prior wetting of the surface with water. The chapter presents degradation experiments with tobacco acid pyrophosphatase. This enzyme degrades pyrophosphate linkages such as those found in ATP or eucaryotic mRNA. As it is known that TAP has difficulty cleaving some pyrophoshate linkages, enough TAP is used to cleave the 5' cap linkage from 3μg of mRNA even though the physical amounts of each TP were below microgram levels. TF–H7 can be radiolabelled with 125 I using the method of Prensky. This finding and the ability to study TF–H7 using PEI–TLC and acrylamide gel electrophoresis open up new frontiers for research on the structure of TF.

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.

A new technique for recombinant cDNA construction is presented in which ribonucleotides are added to the 3′ end of cDNA via ribosubstitution prior to synthesis of the second DNA strand. The hairpin floppy loop is then opened by alkaline hydrolysis. This method eliminates the requirement for S1 nuclease and thus shows promise as a means for preservation of mRNA 5′-end sequences in recombinant cDNAs and for eliminating a potential source of error in those sequences.

Publisher Summary This chapter reviews that cDNA serves as the template for synthesis of a variety of products used in various phases. The synthesis products include 32 P-labeled RNA for sequence analysis of fragments resulting from a cleavage at G residues, 32 P-labeled dC- or dT-substituted RNAs for specific cleavage at U or at C residues, and duplex gene copies for insertion into bacterial plasmids. It reviews that molecular cloning approach is applied to a wide variety of eukaryotic mRNAs. One of the advantages of the technique that is developed for the insertion of eukaryotic mRNA copies into bacterial plasmids is that a rigorous purification is not necessary; the act of cloning such material definitively eliminates all contaminating eukaryotic nucleic acid sequences. This should be especially useful to the researcher who, for instance, has a mixture of 10 different mRNA species that are difficult to separate. To obtain the corresponding sequences in pure form, it suffices to make cDNA copies of the mixture of mRNAs, convert this mixture of cDNAs to duplex gene copies, insert the DNA into bacterial plasmids and introduce the mixture of plasmids thus obtained into bacteria. Since each bacterial transformant can receive only one of the eukaryotic gene inserts, analysis of a few dozen bacterial clones should suffice to provide one with a clone for each of the interesting sequences. With such clones, it is possible to produce milligram quantities of these sequences in duplex form, sufficient for the most rapid sequencing techniques.