A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5′ non‐coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative‐strand RNA genome.

Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the Mononegavirales but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes.

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The amylase complex on mouse chromosome 3 encodes both salivary and pancreatic amylase. It appears that one active gene is present for salivary amylase, whereas pancreatic amylase in some strains is coded by at least 4, and perhaps by more than 10, genes. Strain YBR is different from other strains in that it produces twice as much salivary amylase. Pancreatic amylase in YBR is present as two different protein forms, A beta and B beta, the sum of which amounts to only one-third of that in, for instance, strain A/J. YBR chromosomal DNA was cloned in phage gamma, followed by restriction and heteroduplex analysis of recombinant phages carrying amylase genes. Among 32 phage isolates, 5 carried parts of the salivary amylase sequence. The remaining phage isolates contained pancreatic amylase-like sequences and represented three nonoverlapping genomic regions, i.e., one of 34 kb containing a complete gene, PAN-II beta; another of 41 kb with a complete but different gene, PAN-I beta, plus a truncated gene, PAN-psi 1; and finally, one of 23 kb with another truncated gene, PAN-psi 2. Parts of the amino acid sequence of A beta and B beta have previously been determined, and we report here the sequencing of a 4-kb DNA fragment from Pan-II beta which establishes that this gene codes for B beta.

Background. The distinction of sporadic from inherited medullary thyroid carcinomas (MTCs) is of clinical importance because of the differences in prognosis, and the need for family screening for genetic counseling required in the latter. Germline mutations in the RET protooncogene are associated with multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid carcinoma (FMTC), and MEN type 2B. Somatic point mutations in the same gene have been identified in a subset of sporadically occurring medullary thyroid carcinomas. Methods. A nonisotopic polymerase chain reaction‐(PCR) based single strand conformation polymorphism (SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from 32 form‐aldehyde fixed and paraffin embedded MTC specimens and normal tissue or blood of the same patient for point mutations in RET exons 10, 11, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR‐products using an automated DNA‐sequencer. Results were compared with the disease phenotype, clinical findings, and follow‐up. Results. Six different missense germline mutations were identified at cysteine residues 618, 630, and 634 of the cysteine‐rich extracellular RET domain encoded by exons 10 and 11 in all patients with FMTC and MEN 2A. The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a 634 Cys → Arg mutation was associated with parathyroid disease in three patients. A germline Met → Thr point mutation at codon 918 of the RET tyrosine kinase domain was identified in all three patients with MEN 2B. Two patients with clinically sporadic MTCs and negative family history exhibited a RET germline mutation at codon 634, indicating the presence of an nonpredicted inherited MTC. Further‐more, one patient had a 618 Cys → Ser mutation in the tumor and nontumorous thyroid DNA but not in blood DNA, indicating a mosaic mutation affecting thyroid tissue but not blood cells. Tumor specific (somatic) Met → Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs. The remaining eight sporadic MTCs lacked mutations in all three RET exons tested. Conclusions. This study demonstrates that (1) the molecular methods are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B but also to distinguish heritable from non‐heritable MTCs using archival tissue specimens, and (2) that more MTCs than clinically expected are heritable, indicating the need for genetic analysis of all patients with MTC. Cancer 1995; 76:479–89.