Gamma-irradiation directly affects the formation of coding joints in SCID cell lines.

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.

Osteoclast-like giant cell tumor of the pancreas with metastases to gallbladder and lymph nodes. A case report.

Osteoclast-like giant cell tumor of the pancreas (OGTP) is a rare neoplasm, of which the histogenesis is still controversial. Here we report a case of OGTP involving the head of the pancreas in a 71-year-old woman with metastases to the gallbladder and lymph nodes. The primary and metastatic tumors had identical histopathological, immunohistochemical, ultrastructural and molecular biological features. Microscopically, the tumors were characterized by atypical, often pleomorphic mononuclear cells associated with the proliferation of benign-appearing osteoclast-like giant cells (OGCs). Electron microscopic observation provided ultrastructural evidence of epithelial differentiation of the mononuclear cells, including microvilli and desmosomes, which was not obtained for OGCs. On immunohistochemical study, OGCs stained for CD68 (KP-1), LCA and HAM56, whereas mononuclear cells only reacted with PCNA. These findings clearly suggest that mononuclear cells are capable of differentiation and proliferation and may have been the only true tumor cells in this neoplasm, and that OGCs may have been a paraneoplastic product of this rare tumor. On examination of DNA from dewaxed sections of the tumor, we found no p53 mutation in the tumor tissue, but found two K-ras mutations in codon 12; this pattern of mutation commonly occurs in pancreatic carcinoma, indicating a somewhat genetic relationship of OGTP to pancreatic carcinoma. Although OGTP often has a favorable prognosis, the outcome in the present case was poor due to early tumor spread, with less than two years postoperative survival.

Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.

Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.

The psbC start codon in Synechocystis sp. PCC 6803.

The translation start codon for psbC, the gene encoding CP43, a chlorophyll-binding protein of photosystem II, has been identified for the cyanobacterium Synechosystis sp. PCC 6803 using site-directed mutagenesis. An AUG codon, about 50 bases upstream from the end of psbD-I had previously been assumed to be the translation start site of psbC. However, the fact that the AUG codon is not present in psbC from several other organisms, whereas a GUG codon 14 bases upstream from the end of psbD-I is strictly conserved suggests that CP43 translation starts at the latter codon. Mutation of GUG, but not of AUG, led to a loss of CP43 and photoautotrophic growth, indicating that the GUG codon is the sole initiation site for translation of the CP43 protein in Synechocystis sp. PCC 6803.

The difference between human C3F and C3S results from a single amino acid change from an asparagine to an aspartate residue at position 1216 on the alpha-chain of the complement component, C3.

The third component of the human C system, C3, exhibits two common genetic variants. These variants have been characterized by high voltage agarose electrophoresis and are designated C3 fast (C3F) and C3 slow (C3S). C3F occurs at appreciable frequencies only in Caucasian populations and has been shown to be associated with an increased incidence of certain diseases, such as partial lipodystrophy, IgA nephropathy, and Indian childhood cirrhosis. It has been shown that C3F differs from C3S with regard to isoelectric point as well as its ability to bind macrophages. The availability of a full-length cDNA probe for human C3 has made it possible to study the polymorphism at the genomic level. We have used RNA/RNA hybridization to demonstrate that the difference between C3F and C3S occurs in the C3d region. We subsequently used oligonucleotide-primed DNA amplification to show that C3F arises from a point mutation at codon 1216 converting a deoxyadenosine for a deoxyguanosine. The result of this point mutation at the translational level is the substitution of an asparagine residue in C3S for an aspartic acid residue in C3F. It is known that C3d contains the binding site for CR2 as well as the internal thioester site and multiple protease cleavage sites. The identification of the structural basis of the differences between C3F and C3S will assist our continuing studies of the mechanism of the functional differences between the two alleles and the disease associations of C3F. It also allows us to use DNA based techniques to allotype C3 in subjects with little or no C3 in their serum.

Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency.

Point mutations in the X-linked ornithine transcarbamylase (OTC) gene have been detected at the same Taq I restriction site in 3 of 24 unrelated probands with OTC deficiency. A de novo mutation could be traced in all three families to an individual in a prior generation, confirming independent recurrence. The DNA sequence in the region of the altered Taq I site was determined in the three probands. In two unrelated male probands with neonatal onset of severe OTC deficiency, a guanine (G) to adenine (A) mutation on the sense strand (antisense cytosine [C] to thymine [T]) was found, resulting in glutamine for arginine at amino acid 109 of the mature polypeptide. In the third case, where the proband was a symptomatic female, C to T (sense strand) transition converted residue 109 to a premature stop. These results support the observation that Taq I restriction sites, which contain an internal CG, are particularly susceptible to C to T transition mutation due to deamination of a methylated C in either the sense or antisense strand. The OTC gene seems especially sensitive to C to T transition mutation at arginine codon 109 because either a nonsense mutation or an extremely deleterious missense mutation will result.

Clinical and genetic heterogeneity in black patients with homozygous beta-thalassemia from the southeastern United States.

The presence of various substitutions and deletions resulting in beta-thalassemia was studied in 19 black patients with homozygous beta-thalassemia and in numerous relatives; all patients were from Georgia, South Carolina, and Alabama. Methodology included gene mapping, amplification of genomic DNA with Taq polymerase, identification of known nucleotide substitutions or a single nucleotide deletion through hybridization with synthetic oligonucleotides, cloning and sequencing of a beta-globin gene, and sequencing of amplified genomic DNA. Of the 38 chromosomes tested, 21 (55%) had the A----G substitution at nt -29, eight (21%) had the C----T substitution at nt -88, three (8%) had the substitution at codon 24, while one each of the following abnormalities were also detected: frameshift at codon 6, a C----A mutation at nt 848 of the beta IVS-II (new), an A----T mutation at codon 61 (new), a deletion of 1.35 kilobases including the 5' end of beta, a Ggamma(Agamma delta beta)(0)-thalassemia, and one thalassemia determinant that remained unidentified. The C----A mutation at nt 848 of IVS-II occurred at a position 3 nucleotides 5' to the third exon, adjacent to the invariant AG dinucleotide of the acceptor sequence. The A----T mutation in codon 61 (AAG----TAG) resulted in the creation of a stop codon and thus in beta(0)-thalassemia. The various mutations occurred on chromosomes with different haplotypes; however, chromosomes with a specific mutation but with different haplotypes belonged to one specific framework, which suggested that crossovers were responsible for these different types. Hemoglobin (Hb) F levels were generally high (55% to 75% with 98.5% in one patient with beta(0)/beta(0)); a few patients with specific haplotypes and an alpha-thalassemia-2 heterozygosity had a lower Hb F level. The Ggamma in the Hb F was consistently high when the C----T mutation occurred at nt -158 to the Cap site of the Ggamma-globin gene; seven patients with +/+ at this site had an average Ggamma of 73.8%, eight patients with +/- had 64.8%, and one patient with -/- had 34.2%. Variations in hematologic values and in Hb F, Ggamma, and Hb A2 levels of relatives with a beta-thalassemia heterozygosity depended to some extent on the types of mutations or deletions and on the haplotypes of the chromosomes with the beta-thalassemia determinant.

KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas.

DNAs from human pancreatic adenocarcinomas were analyzed for the presence of mutations in codons 12, 13 and 61 of the NRAS, KRAS and HRAS gene. Formalin-fixed and paraffin-embedded tissue was used directly in an in vitro amplification reaction to expand the relevant RAS sequences. The mutations were detected by selective hybridization using mutation-specific synthetic oligonucleotides. In 28 of the 30 patients we found a mutation in codon 12 of the KRAS gene. This result confirms the findings of Almoguera et al. [Cell 53 (1988) 549-554] that KRAS mutations occur frequently in adenocarcinomas of the exocrine pancreas. The mutations are predominantly G-T transversions, in contrast to the KRAS mutations in colon tumors which are mainly G-A transitions. Furthermore, in a portion of the tumors the mutation appears to be homozygous.

c-K-ras codon 12 GGT-CGT point mutation. An infrequent event in human lung cancer.

Hu-c-ras represent a family of oncogenes which are capable of inducing malignant transformation in the NIH/3T3 mouse cell line. Associated with this transformation are specific point mutations observed in the 12th and 61st codon of c-K-ras and N-ras and c-Ha-ras, respectively. These base changes generate, in some instances, a new restriction enzyme cleavage site and a restriction fragment length polymorphism (RFLP). One such RFLP has recently been reported for the mutation GGT-CGT at codon 12 of c-K-ras. Our data suggest that this point mutation is rarely present in human lung cancer and therefore is not likely to play a major role in cancer development.

lacZ--Y+ fusions in Escherichia coli. DNA sequencing reveals the eight N-terminal residues of lac permease as non-essential.

The nucleotide sequence of three lacZ- -Y+ fusions found among spontaneous lacY+ revertants of the lacY- mutant MAB16 in Escherichia coli is reported. MAB16 is a frameshift mutation in codon 6 of the lacY gene. DNA sequence analysis of the fusions shows the first eight N-terminal codons of the lacY gene can be replaced by 3, 39 or 804/805 N-terminal codons of the lacZ gene without impairing lac permease activity qualitatively.