A deficiency spanning section 84B-D of the proximal right arm of chromosome 3 of Drosophila melanogaster has been utilized as a screen for mutations that aid in the genetic dissection of this developmentally interesting region.-Ninety-two mutations have been recovered, of which 12 have been localized by deficiency and complementation mapping to the 84B1,2 doublet, the site of the Antennapedia complex (ANT-C). This has permitted a more precise description of the genetic organization of this complex locus.-A collection of 31 mutations that reside in 84C1-D2 displays an intricate circular complementation pattern, characterized by two clusters of mutations that exhibit semi-lethality, negative complementation and temperature sensitivity. Mutations affecting seven additional functional groups within the 84B-D region were also recovered.

Temperature-sensitive mutations fall into two general classes: those generating thermolabile proteins; and those generating defects in protein synthesis, folding or assembly. Temperature-sensitive mutations at 17 sites in the gene for the coat protein of Phage P22 are of the latter class, preventing the productive folding of the polypeptide chain at restrictive temperature. We show here that, though the coat subunits interact intimately to form the viral shell, these temperature-sensitive folding (TSF) mutations were all recessive to wild type. The mutant polypeptide chains were not rescued by the presence of wild-type polypeptide chains. Missense substitutions in multimeric proteins frequently exhibit intragenic complementation; however, all pairs of coat protein TSF mutants tested failed to complement. The recessive phenotypes, absence of rescue and absence of intragenic complementation are all accounted for by the TSF defect, in which destabilization of a folding intermediate at restrictive temperature prevents the mutant chain from reaching the conformation required for subunit/subunit recognition. We suggest that absence of intragenic complementation should be a general property of TSF mutations in genes encoding multimeric proteins. The spectra of new loci identified by isolating second-site suppressors and synthetic lethals of temperature sensitive mutants will also differ depending on the nature of the defect. In the case of TSF mutations, where folding intermediates are defective rather than the native molecule, the spectra of other genes identified should shift from those whose products interact with the native molecule to those whose products influence the folding process.

As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.--None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of these ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.

In this paper, we present results of crosses designed to elucidate the structure of recombinants in the tail-fiber region of bacteriophage T4, in which a glucosylation-dependent recombinations mechanism is operative, and the cause of the "special" recombination in glycosylated crosses is discussed. We present evidence that, when phage are nonglycosylated, recombination in the tail-fiber region proceeds via long heteroduplex overlaps. Mismatched bases within such regions (in nonglycosylated phage) are repaired efficiently (as contrasted to those of glucosylated phage), but asymmetrically; that is, there may be an equal probability of resolving the mismatch to mutant or wild type.

Interallelic complementation between certain temperature-sensitive mutants of gene 42 of bacteriophage T4 was demonstrated by measuring the incorporation of labeled thymine into DNA.

Following infection of E. coli B with ligase-deficient rII bacteriophage T4D recombination between linked markers is increased 4.2 fold and heterozygote frequency increased 2.3 fold. In such infection recombination occurs at a rapid rate for an extended period. This is in contrast to the time course of recombination observed in wild-type, lysis-inhibited, or lysis-defective (gene t defective) infection. In all of these cases recombination under standard cross conditions occurs early in the vegetative cycle. The increased recombination in ligase-deficient rII infection is reduced in a bacterial strain which produces greater than normal levels of host ligase. These results indicate that ligase has a crucial role not only in the replication of DNA but also in recombination. The level of ligase may determine whether DNA replication occurs with or without concomitant recombination.

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.

Some new, generally nonchemotactic mutants of Salmonella typhimurium were isolated and they, together with previously isolated mutants and some from other investigators, were mapped. Most of the mutants were classified in nine complementation groups, which are probably individual genes. Of these, five map at the end of the flagella region and appear in the order motB-(cheWcheP)-cheX-cheQ-cheR-flaC. Two of the mutations, cheU and cheV, map in the flaQ and flaAII genes, respectively. The remaining genes, cheS and cheT, have not yet been mapped. Most of the mutants are phenotypically smoothly swimming, but some are constantly tumbling. Two of the groups show dominant behavior as recipients in genetic crosses; the rest are recessive. The mutants vary in their responses to stimuli but, since their responses to all chemoeffectors are abnormal, the central processing, rather than individual, receptors must be impaired. The two mutations that coincide with genes for flagella probably involve the locus of the final delivery of sensing signal to the flagella.