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In an attempt to characterize the genes that cause immunodeficiencies such as X-linked agammaglobulinemia (XLA) and severe combined immunodeficiency (SCID) we established precursor B-cell lines by transforming the patients' bone marrow cells with Epstein-Barr viruses. DNA rearrangements of immunoglobulin JH gene loci were observed on both chromosomes in pre-B cells derived from an XLA patient. We cloned and characterized both rearranged bands from one cell line. Both of the rearrangements occurred between DH and JH gene loci without the VH-DH structure. On the other hand, JH gene loci retained the germline configuration on both chromosomes in almost all the transformants derived from a SCID patient that had been determined according to their surface markers, to be in an early precursor B-cell stage. The implications of the observations are discussed.

The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9-kb repeating sequences. We determined the nucleotide sequence of a 15-kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9-kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12-nucleotide spacer signals. Most of the somatic DH sequences found in the published VH-DH-JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR-DH or DH-DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.

cDNA of rat pepsinogen C (PGC) hybridizes to, among others, a 3.2-kb band in Southern blot analysis of BamHI-cleaved human genomic DNA. This property was employed to localize the human PGC gene. Use of flow-sorted human chromosomes and 12 human x mouse somatic cell hybrid lines demonstrated that the gene is located on chromosome 6.

A nearly full-length rat pepsinogen cDNA was isolated from a rat gastric mucosa cDNA library and its nucleotide sequence was determined. The cDNA comprises 1370 base pairs (bp), including the 5'-non-coding region (60 bp), the coding nucleotide sequence (1176 bp) and the 3'-non-coding region (131 bp). The predicted amino acid sequence of rat prepepsinogen (392 residues) contains a 16-residue signal sequence followed by the pepsionogen moiety of 376 residues. Rat pepsinogen has an amino acid composition characteristic of C-type pepsinogens and is much more homologous in amino acid sequence with C-type pepsinogens than A-type pepsinogens. These results indicate that the major form of rat pepsinogen can be classified as a C type pepsinogen.

Both primates and rodents apparently originated from insectivores and then evolved separately. We isolated the immunoglobulin mu gene from DNA of the insectivore Suncus murinus and determined its nucleotide sequence. The gene organization was CH1 exon (318 bp)-intron (89 bp)-CH2 exon (345 bp)-intron (80 bp)-CH3 exon (318 bp)-intron (85 bp)-CH4 exon (392 bp)-coding sequence and 3'-untranslated region. Comparison of nucleotide sequences of mu genes between mouse, human and Suncus murinus indicated that the evolutionary distance between human and mouse is equal to that between Suncus murinus and human, and that mouse is deviated further from Suncus murinus than the two other combinations. This conclusion was further supported by sequence comparison of non-coding regions.

The entire human pepsinogen C gene has been isolated from a cosmid genomic library. The nucleotide sequences of all the exons and the 5'- and 3'-flanking regions of the gene are presented. The organization of the gene is fundamentally compatible with those of other aspartic proteinases, allowing us to conclude that the genes of these aspartic proteinases including pepsinogen C are derived from a common ancestral gene. The predicted 388-residue amino acid sequence of human pepsinogen C consists of a signal sequence of 16-amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two active-site aspartic acids. In the light of present notions about eukaryotic gene expression, possible regulatory roles of the oligonucleotide DNA sequences in the promoter region of the gene are discussed.

The pepsinogen gene is hypomethylated in the stomach, in which it is expressed. For demonstration that this hypomethylation of the pepsinogen gene in the stomach reflects pepsinogen-producing cells, we analyzed fractions of dispersed mucosal cells with various contents of pepsinogen-producing cells prepared from guinea pig stomach by centrifugal elutriation. mRNA expression and the extent of hypomethylation of the pepsinogen gene in each fraction was closely correlated with the content of pepsinogen-producing cells. These results suggested hypomethylation of the pepsinogen gene in pepsinogen-producing cells and differential pepsinogen gene methylation in cell subpopulations in the stomach.

The major translation product of rat gastric mucosa RNA in a wheat germ cell-free system was identified as prepepsinogen by electrophoretic analysis of its immunoprecipitate on sodium dodecyl sulfate (SDS)-polyacrylamide gels and amino-terminal sequence determination. The translation product containing radioactive amino acids, purified by SDS-polyacrylamide gel electrophoresis, was shown to have an amino-terminal extension peptide comprising 16 amino acid residues. A partial amino acid sequence of this extension peptide is as follows: Met-X-X-Met-Val-Val-X-Leu-Leu-X-Leu-X-Leu-Leu-X-X-pepsinogen.

The entire rat pepsinogen C gene has been isolated from a rat genomic library, using the rat pepsinogen C cDNA as a probe. Southern blot analysis showed that there exists at least two rat pepsinogen C genes. The nucleotide sequences of the coding regions and the 5'- and 3'-flanking regions of one of the rat pepsinogen C genes have been determined. This gene is split into 9 exons interrupted by eight intervening sequences. The 5'-flanking region is similar to that of the human pepsinogen C gene, but only the former has the core sequence of the Sp1 binding site. The amount of transcripts of the rat pepsinogen C genes was found to increase during development, and a similar increase was shown to be induced by injection of hydrocortisone. As a candidate of a factor which regulates the transcription, we found a 25-kDa protein by Southwestern blotting. It binds to a specific site in the 5'-flanking region of the gene only in the presence of Mg2+ ion, and it is present in the nuclear fraction of the gastric mucosa but not of the liver.

The total RNAs were extracted from human, swine, rat, and calf gastric mucosae, and translated in vitro in the presence of radiolabeled amino acids using a wheat germ cell-free system. Upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the translation products, a protein band with a molecular weight of about 43,000 was obtained in each case as one of the major products. These products could be specifically immunoprecipitated with a corresponding anti-pepsinogen or anti-chymosin antiserum. Radiosequence analysis of these translation products purified by SDS-polyacrylamide gel electrophoresis showed that each of them is a precursor form, i.e., prepepsinogen or preprochymosin, having an amino-terminal extension peptide (signal sequence) comprising 15 (human and swine) or 16 (rat and calf) amino acid residues. The primary structures of these signal sequences were determined to be as follows: (Sequence: see text). These signal sequences share common characteristics with those of other pre-secretory proteins, i.e., the presence of positive charges in the NH2-terminal region, hydrophobic amino acid clusters in the interior part, and amino acids with short side chains at the site of cleavage by the signal peptidase.

The variable region of immunoglobulin (Ig) heavy chain is encoded by three separate genes: variable (VH), diversity (DH) and joining (JH) genes on the germ-line genome. In mice, most complementarity determining region (CDR) III of the heavy chains of myelomas and hybridomas sequenced so far can be assigned to one of the 12 already identified germ-line DH genes by the homology of nucleotide sequences of DH gene-coding regions although extranucleotides, the so-called N segments, are found at the boundaries between DH and JH as well as VH and DH. On the other hand, Siebenlist et al. (Nature 1981. 294:631) identified two DH gene families in human genome: DHQ52, located at 45 bp upstream of the JH gene cluster, and another family encoded at 9-kb regular intervals possibly between VH and JH gene clusters. However, the somatic DH sequences found in VH-DH-JH structure (the somatic DH segment being defined as the region which is not encoded either by germ-line VH or JH gene) are relatively long and apparently random, and do not seem to have the homology to any of the germ-line DH sequences. To explain the origin of high diversity in the CDR III of human Ig heavy chains, Siebenlist et al. predicted the presence of another mechanism, namely DH-DH joinings. In the present study, we identified five DH genes in one of the above 9-kb repeats. This suggests that the total number of germ-line DH genes is much higher in man than in mouse. The comparison between somatic DH sequences and germ-line DH sequences indicates that most somatic DH sequences in human Ig heavy chains are also produced by VH-DH and DH-JH joinings without the joining of multiple DH gene segments.

A simple and sensitive method for proteinase assay was developed, which uses fluorescamine-labeled casein-Sepharose 4B as a substrate. Casein-Sepharose 4B was prepared most effectively by coupling casein to cyanogen bromide-activated Sepharose 4B at pH 10.0. Fluorescamine-labeled casein-Sepharose 4B was then prepared by mixing fluorescamine and casein-Sepharose 4B suspension at pH 8.0 and used for the assay as a substrate after removal of the excess reagent and/or its hydrolysis products. The assay can be done by simple measuring the fluorescence (excitation at 390 nm and emission at 475 nm) of the filtrate of the assay mixture after incubation of the substrate with enzyme solution. This method is suited for the assay of proteinases active at neutral to slightly alkaline pH values, and the activity of 3 ng of trypsin or 10 ng of alpha-chymotrypsin can be determined with reasonable accuracy. This method is therefore almost as sensitive as those using radioisotope-labeled proteins as substrates.

Three new furanoditerpenyl glucosides, columbinyl glucoside, jateorinyl glucoside, and isojateorinyl glucoside were isolated from JATEORRHIZA COLUMBA roots. Their (13)C-NMR chemical shifts were assigned.

A calcium-activated neutral protease (CANP) was purified from monkey cardiac muscle by a method involving column chromatography on DEAE-cellulose, Sepharose CL-6B, DEAE-Sephacel, organomercurial-Sepharose 4B, and Sephadex G-150 in succession. This protease required both millimolar concentration of Ca2+ and the SH-group for activation, and it was maximally active around pH 8.0. It was strongly inhibited by thiol protease inhibitors such as iodoacetic acid, antipain, leupeptin, and epoxysuccinic acid derivatives. The molecular weight of this protease was estimated to be 110,000 by gel filtration. Upon nondenaturing electrophoresis the purified protease gave two bands, both of which were active at millimolar concentration of Ca2+, indicating the existence of two forms of the protease. The less acidic band (form I CANP) contained two components with molecular weights of 74,000 and 28,000 and the more acidic one (form II CANP) contained components with molecular weights of 74,000 and 26,000. The protease was synergistically activated by Mn2+ and Ca2+ at a concentration where Mn2+ or Ca2+ alone was not effective. In the presence of millimolar level of Ca2+, limited autolysis reduced the Ca2+-requirement of this protease. The proteolysis of myofibrils by this protease resulted in the production of a component with a molecular weight of 30,000 as well as various other higher and lower molecular weight peptide fragments.

The relationship between methylation and expression of rat pepsinogen 1 (Pg1) genes was investigated in various tissues. On Northern blotting with a Pg1 complementary DNA probe, Pg1 mRNA was detected only in the glandular stomach of normal rats. Methylation analysis with Msp1/HpaII and Hha1 revealed tissue specific methylation patterns of Pg1 genes with less methylated in the stomach than in other normal tissues not expressing the genes. During stomach development, there was a progressive increase in the Pg1 mRNA level that almost coincided with change in the mucosal pepsinogen level and progressive demethylation after the onset of transcription. Thus, there was an inverse correlation between methylation and expression of Pg1 genes, suggesting a role of DNA methylation in Pg1 gene regulation during normal differentiation, although not its primary role in gene activation. There was no detectable Pg1 mRNA in either primary or transplanted stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine. The methylation patterns of Pg1 genes were different from those of normal tissues that expressed the gene and of those that did not and no simple correlation was observed between methylation and expression of Pg1 genes. This result is consistent with a previous finding that DNA methylation is deranged in tumor cells.

A recombinant clone, which covers the pepsinogen gene in a single insert, has been isolated by screening a library of human genomic DNA, using a swine pepsinogen cDNA as a probe. Sequence analysis of coding DNA segments of the clone revealed that the pepsinogen gene occupies approximately 9.4-kilobase pairs of the genomic DNA and is separated into nine exons by eight introns of various lengths. The predicted amino acid sequence of human pepsinogen consists of 373 residues and is 82% homologous with that of swine pepsinogen. In addition, the predicted sequence contained a single sequence of 15 amino acid residues at the NH2 terminus, showing that the protein is synthesized as prepepsinogen. The structure of the gene, in which two homologous sequences including the two active site aspartyl residues of pepsin are present in different coding segments, is in support of the view that the pepsinogen gene evolved by duplication of a shorter ancestral gene.

We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al.

The extract of seeds of Euphorbia lathyris L. showed antitumor activity against Sarcoma 180 ascites in mice. Systematic fractionation of the extract led to the characterization of ingenol-3-hexadecanoate as an active principle, together with an inactive diterpene ingenol-20-hexadecanoate.