The metabolism of macromolecules during the differentiation of myxamoebae of the cellular slime mould Dictyostelium discoideum containing different amounts of glycogen

1. Methods of obtaining myxamoebae of Dictyostelium discoideum strain Ax-2 (ATCC 24397) with glycogen contents in the range 0.05-5mg of glycogen/10(8) cells are described. The changes in cellular glycogen, protein and RNA content during the differentiation of such myxamoebae were determined. 2. Myxamoebal glycogen is not conserved during differentiation and gluconeogenesis may occur even in cells that contain a large amount of glycogen initially. 3. There is a marked net loss of cellular protein and RNA during differentiation and associated with this there are also marked decreases in the sizes of the intracellular pools of amino acids, acid-soluble proteins and pentose-containing materials. 4. During the early stages of development some protein and pentose(s) are excreted, but this cannot account for the decreased cellular content of protein and RNA. 5. There is a linear rate of production of NH(3) during development, and oxidation appears to be the fate of the major portion of the degraded protein and RNA. 6. However, provision of an alternative metabolizable energy source (glycogen) has little effect on the rate or extent of protein or RNA breakdown or on the changes in the sizes of the intracellular pools of amino acids, acid-soluble proteins and pentose-containing materials. 7. It is concluded that during development there is a requirement for the destruction of specific RNA and protein molecules for reasons other than the provision of oxidizable substrates. 8. The kinetic model of Wright et al. (1968) is discussed in relation to these changes in macromolecular content.     

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.     

Inheritance of quantitative expression of erythrocyte glucose-6-phosphate dehydrogenase activity in the Negro—a twin study

Studies have been conducted on eight sets of monozygous and nine sets of dizygous female Negro twins, both members of whom were heterozygous for G-6-PD deficiency. Twins were studied both by assay of erythrocytic G-6-PD activity and by the methemoglobin elution test (MET). The MET is a procedure which identifies histochemically cells with appreciable G-6-PD activity and permits accurate determination of the percentage of such cells in heterozygotes. Monozygous twins showed significantly less within-pair variation than dizygous twins with both the MET and G-6-PD assay.Concerning the significantly greater agreement in MET results in monozygous twins than dizygous twins, our present working hypothesis is that X-chromosomal inactivation in the Negro female is genetically controlled, rather than random. However, certain alternate hypotheses allowing for random X-inactivation have not been excluded; these include somatic cell selection after random X-inactivation, and cell exchange between identical twins in utero/it. Studies in nontwin related heterozygotes now underway should help differentiate among these various possibilities.In addition to the studies on 17 pairs of female twins heterozygous for G-6-PD deficiency, 26 pairs of nondeficient female Negro twins have been studied by G-6-PD assay. Within-pair variation in monozygous twins was significantly less than within-pair variation in dizygous twins in all cases. The genetic influences detected with the G-6-PD assay in the female twins could theoretically be due to nonrandom X-inactivation, to genetically determined quantitative differences in enzyme activity (e.g., isoalleles), or to both. By appropriate calculations, based on the MET results, we have factored out the effects of X-inactivation on overall enzyme activity in the heterozygous deficient twins. After removal of the effect of X-inactivation, monozygous twins heterozygous for enzyme deficiency continue to show significantly less within-pair variation than dizygous twins. This finding indicates significant genetic influences on quantitative G-6-PD activity other than X-inactivation and other than the deficiency allele. This conclusion has been strengthened by studies on male twins where X-inactivation is not present.Supported by USPHS research grants AM-09381, HE-17544, AM-09919, and HE-03341, by USPHS Career Development Award 1-K3-AM-7959 (Dr. Brewer) and by U.S.A.E.C. Contract (11-1)-1552.     

Multiple-mutation reaction: a method for simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene.     

A Survey of Non-Classical Polyandry

We have identified a sample of 53 societies outside of the classical Himalayan and Marquesean area that permit polyandrous unions. Our goal is to broadly describe the demographic, social, marital, and economic characteristics of these societies and to evaluate some hypotheses of the causes of polyandry. We demonstrate that although polyandry is rare it is not as rare as commonly believed, is found worldwide, and is most common in egalitarian societies. We also argue that polyandry likely existed during early human history and should be examined from an evolutionary perspective. Our analysis reveals that it may be a predictable response to a high operational sex ratio favoring males and may also be a response to high rates of male mortality and, possibly, male absenteeism. Other factors may contribute, but our within-polyandry sample limits analysis.