Expression of mitochondrial DNA in Saccharomyces cerevisiae: the construction of sets of isonuclear haploid strains containing different specified mitochondrial genomes.

In order to perform systematic biochemical studies of the expression of the mitochondrial genome in different strains of $\text{Saccharomyces}\text{cerevisiae}$, we have constructed a series of haploid strains in which a specified mitochondrial genome has been placed in cells containing a particular nuclear genome. The desired strains were obtained from crosses between $\text{rho}{}^{\mathrm{+}}$ and $\text{rho}{}^{\mathrm{o}}$ haploids, one of which carried the $\text{karl}$-l mutation which interferes with nuclear fusion. These newly constructed strains provide the basis for studying mitochondrial gene expression as a function of (i) the nucleotide sequence differences that are apparent in the mitochondrial DNAs from different strains, and (ii) the role played by different nuclear genomes in modulating the expression of mitochondrial genes.     

Preliminary x-ray diffraction studies on an anti-viral protein.

An anti-viral enzyme from $\text{Phytolacca americana}$, known to inhibit protein synthesis has been crystallized in a form useful for high resolution x-ray diffraction studies. Cracking of crystals due to the introduction of heavy metals can be reduced by cross linking with glutaraldehyde using a rapid fixation method. Several apparent isomorphous heavy metal derivatives of the crystal have been found. The molecular weight of the protein has been reevaluated as 31,000 daltons.     

Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects.

Proctolin, a myotropic substance with potent activity on the proctodeal (hindgut) muscles of the cockroach, $\text{Periplaneta americana}$ (L.), has been identified by the use of Edman degradation and dansylation techniques as Arg-Tyr-Leu-Pro-Thr. Synthesis of the pentapeptide having this sequence and exhibiting the properties of natural proctolin confirmed the structure. Threshold activity on proctodeal muscle occurs at about 10⁻⁹ M proctolin.     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

Studies of the genetic and immunologic components of the maternal age effect

To assess the genetic component of the maternal age effect, the frequencies of aneuploid embryos derived from several inbred mouse strains were examined as a function of maternal age. Some of the strains were chosen to evaluate possible etiologic agents: CBA for premature reproductive aging and increased univalent frequency, and $\text{NZB}\text{J}$ and $\text{A}\text{J}$ for predisposition toward autoimmunity. The $\text{C3H}\text{HeJ}$ and $\text{C57BL}\text{6J}$ strains also were examined. Although there was considerable variation between strains, an increased frequency of aneuploid fetuses was observed in all strains as a function of maternal aging. The CBA strain revealed the greatest increase in frequency of aneuploid fetuses at a relatively early maternal age. The effect of autoimmunity was assessed by analyzing the frequency of aneuploid embryos derived from thymectomized $\text{A}\text{J}$ females. No significant difference in aneuploidy was found between thymectomized and control nonthymectomized animals.     

Trypanosoma vivax: Courses of infection with three stabilates in inbred mouse strains

The courses of infection in inbred mouse strains were compared following infection with three Stabilates of high, intermediate, and low virulence of Trypanosoma vivax stock Zaria Y486. Mouse strains could only be shown to differ in their resistance to T. vivax infections as judged by the height of the initial parasitemia and survival times when a trypanosome population of low or intermediate virulence was used. A T. vivax population of high virulence was uniformly lethal. Comparison of lytic antibody titers between groups of resistant ($\text{C}\text{57}\text{B}\text{1}\text{6}$) and susceptible ($\text{Balb}\text{c}$) mice did not show any significant differences in titers of the surviving mice but the mice in either group which did not control the initial parasitemia had lower lytic antibody titers than those which did. A significantly larger number of $\text{Balb}\text{c}$ mice failed to control the initial infection as compared to the $\text{C}\text{57}\text{B}\text{1}\text{6}$. Treatment with cyclophosphamide did not ablate differences in susceptibility between the two strains. The use of congenic mice showed that these differences in susceptibility were not related to differences in the major histocompatibility complex between these strains.     

Occurrence of proteinase a isoinhibitors in wild type yeast strains and commercial baker's yeast

The occurrence of the proteinase A inhibitors 2 and 3 was investigated in wild type strains of $\text{Saccharomyces}\text{cerevisiae}$ and $\text{Saccharomyces}\text{carlsbergensis}$ as well as in several strains of commercial baker's yeast. Haploid and diploid strains of $\text{Saccharomyces}\text{cerevisiae}$ contain only proteinase A inhibitor 3 whereas in $\text{Saccharomyces}\text{carlsbergensis}$ only proteinase A inhibitor 2 is found. Strains of commercial baker's yeast contain either proteinase A inhibitor 3 or both inhibitors in a constant ratio of 1:3. Single cell cultures isolated from a strain of commercial baker's yeast also contain a mixture of the two inhibitors. Therefore, baker's yeast is not a mixture of two different cell types but the genome for both inhibitors is present in each single cell. In general, the results indicate that the occurrence of the two proteinase A inhibitors is determined genetically and, therefore, they may be called “isoinhibitors”.     

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ($\text{Nicotiana tabacum}$ cv. Wisconsin #38) crown gall strains incited by $\text{Agrobacterium tumefaciens}$ C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl-$\text{trans}$-zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.     

Role of the mitochondrial protein synthesis is the catabolite repression of the petite-negative yeast K.lactis.

The effect of glucose in two different strains of the petite-negative yeast $\text{K. lactis}$ is studied. The results obtained show that one strain ($\text{K. lactis}\text{CBS 2359}$) is glucose repressible for Glutamate Dehydrogenase and β-Galactosidase, whereas the other one (CBS 2360) is almost completely insensitive. The effect of Erythromycin on expression of catabolite repression in CBS 2359 is also analyzed. The results show that the dependence of catabolite repression on mitochondrial protein synthesis reflect the degree of interaction between the nuclear and mitochondrial compartments.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

Characterization of DNA from Rhizobium cells and their bacteriods from root nodules

DNA from two rhizobial strains $\text{Cicer}$ and $\text{Phaseolus}$ and their bacteroids from root nodules have been isolated, purified and characterized for thermal denaturation temperature and buoyant density. Bacteroid DNA had a lower Tm value and buoyant density comparad to $\text{Rhizobium}$ cell DNA. The calculated GC content of becteroid DNA was lower than the $\text{Rhizobium}$ cell DNA.     

Alteration in molecular species of phosphatidylethanolamine between anesthetic resistant and sensitive strains of drosophila melanogaster

An analysis of lipid composition was carried out in resistant and sensitive strains of $\text{Drosophila}$$\text{melanogaster}$. Amount of total lipid and amount of phosphate of phospholipids were not different from each other in both strains. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) did not differ in amount between both strains. Determination of molecular species of PE using gas chromatography (GC) and GC-mass spectrometry showed that the resistant strain had increased 34:2 and decreased 36:2, 36:3 and 36:5 relative to the sensitive strain. The molecular species of PC did not differ between the two strains. Chromosomal analysis revealed that the alterations in 34:2 and 36:2 of PE were regulated by the X- and third chromosomes of the resistant strain. Therefore, the changes in PE may explain the mechanism of anesthetic resistance because genetic analyses indicate that these chromosomes have more influence on the anesthetic resistant traits of the resistant strain.     

Transposon (Tn5)-mediated suppressive integration of ColE1 derivatives into the chromosome of Escherichia coli K12 (dnaA)

While integration of ColE1 had not been observed previously by ordinary suppressive integration, a $\text{dnaA}$ (Ts) $\text{E. coli}$ strain with Tn$\text{5}$ at various sites of the chromosome and ColE1 or its mini-derivative, pAO3, but not pSC101, inserted by the same transposon produced integratively suppressed strains depending on the RecA function. In contrast to Hfr strains made with a stringently controlled plasmid, they contained the plasmid not only in an integrated but in an autonomous state at an amount comparable to the strain containing the plasmid only autonomously. Introduction of a RecA-deficient mutation to the strain with an integrated ColE1 derivative through conjugation failed. This is likely to be due to lethality of such a strain without RecA-dependent excision of the integrated high copy number plasmid or to quantitative deficiency of DNA polymerase I in addition to the $\text{recA}$ mutation.     

Conversion of a powerful frameshifter acridine to a base-pair substitution analog.

The frameshift mutagenic mechanism for acridines has been attributed to the intercalative type of association between acridines and nucleic acids. However, it appears that these molecular details are insufficient to explain the frameshifting process. In order to design an effective drug probe to analyze the $\text{in vivo}$ interactions of acridines leading to frameshifting, an azide analog of 9-aminoacridine was studied in Ames' $\text{Salmonella}$ strains. The surprising findings were that by substituting an amino group at the 9 ring position with an azido group, the mutagenicity was converted from frameshifter to base-pair substitution.     

Hymenolepis nana: Immunogenicity of a lumen phase of the direct cycle and failure of autoinfection in BALBc mice

When $\text{BALB}\text{c}$ mice were given $\text{BALB}\text{c}$ mouse-derived cysticercoids (cysts) of Hymenolepis nana, only $\text{1}\text{43}$ mice became autoinfected, whereas most ($\text{31}\text{38}$) of dd mice given the same infection became massively autoinfected with mature worms. When $\text{BALB}\text{c}$ mice initially given cysts were challenged with eggs on Day 7, just before the patency of the primary infection, there was normal development into cysts, but almost none of them developed into adult worms. Thus, the failure of autoinfection of H. nana in $\text{BALB}\text{c}$ mice was not a result of failure of eggs to differentiate into cysts in the intestinal tissue, but a result of failure of these cysts to develop into adult worms in the lumen. The reasons why autoinfection does occur in dd and other strains of mice and not in the $\text{BALB}\text{c}$ strain are discussed in terms of the difference in onset of the late response in these strains of mice, ie., the response that is acquired after egg inoculation, and is directed against the lumen phase of cyst challenges. It is strongly suggested that (1) the lumen phase which follows cyst inoculation is highly immunogenic, but clearly differs from tissue phase which follows egg inoculation, (2) the autoinfection which occurs in some strains of mice is therefore not a result of no or poor immunogenicity of the lumen phase but is due to a delay of onset of the late response with the result that a secondary generation may mature, and (3) in other strains of mice, including $\text{BALB}\text{c}$, which acquire the late response within 15 days of initial egg inoculation, autoinfection normally does not occur after cyst infections.     

Coliphage T7 receptors are present in Pseudomonasaeruginosa rough lipopolysaccharides

Lipopolysaccharides (LPS) isolated from various rough derivatives of $\text{Pseudomonas}\text{aeruginosa}$ strains were found to neutralize coliphage T7. Concentrations of 0.4 – 17 μg LPS/ml were sufficient for 50% inactivation of T7 within 1 hour. From the LPS analyses of the mutants, it is believed that T7 may be binding to the heptose region of $\text{P.}\text{aeruginosa}$ LPS, suggesting a similarity in structure between the heptose regions of $\text{P.}\text{aeruginosa}$ and $\text{Escherichia}\text{coli}$ LPS.     

Chloramphenicol damages bacterial DNA.

L(+)-$\text{threo}$-chloramphenicol induces reversion of His^?$\text{Salmonella typhimurium}$ strains TA100 and TA1535 in the conventional Ames' assay without microsomal activation. Any mutagenicity of D(?)-$\text{threo}$-chloramphenicol was masked by toxicity. Similarly, a sensitive fluctuation test showed mutagenesis with L(+)-$\text{threo}$-chloramphenicol at concentrations of 0.5 μM and above but the D(?) isomer proved to be toxic even at these low levels. The L(+) isomer caused single strand breaks in the DNA of $\text{Escherichia coli}\text{B}\text{r}$ and $\text{Salmonella typhimurium}$ strains TA1535, TA100 and TA1976. The D(?) isomer caused breaks in $\text{Escherichia coli}\text{B}\text{r}$ and $\text{Salmonella typhimurium}$ TA1976 although it was less effective and it did not produce DNA breaks in TA1535 or TA100.     

Use of lac operon fusions to isolate Escherichia coli mutants with altered expression of the fumarate reductase system in response to substrate and respiratory controls.

Strains of $\text{E}\text{.}\text{coli}$ with fusions between the $\text{lac}$ structural genes and the promoter region of the fumarate reductase system were constructed from a parental strain deleted in the native $\text{lac}$ operon. Like fumarate reductase in wild-type cells, β-galactosidase in these fusion strains is inducible by fumarate, but only under anaerobic conditions. From one of these strains, three classes of mutants altered in the expression of the hybrid operon were isolated. By anaerobic selection for growth on lactose in the absence of fumarate, mutants that synthesize β-galactosidase constitutively both aerobically and anaerobically were obtained. By aerobic selection for growth on lactose in the presence of fumarate, mutants that are inducible in the enzyme both aerobically and anaerobically and mutants that are inducible in the enzyme only aerobically were obtained. The regulatory behaviors of the mutants studied suggest that substrate and respiratory control of the expression of the fumarate reductase complex are mechanistically connected.