Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Can transposable element copy number distribution parameters be estimated from natural populations of Drosophila melanogaster?

The copy frequency distribution of a transposable element family in a Drosophila melanogaster natural population is generally characterised by the values of the Charlesworths' model parameters α and β (Charlesworth & Charlesworth, 1983). The estimation of these parameters is made using the observed distribution of the occupied sites in a population sample. Several results have been interpreted as due either to the influence of stochastic factors or to deterministic factors (transposition, excision, selection…). The accuracy of this method was tested by estimations performed on samples from simulated populations. The results show that with the sample size usually used for natural population studies, the confidence intervals are too large to reasonably deduce either the element copy number distribution or the values of transposition and excision rate and selective coefficients.     

Body size but not colony size increases with altitude in the holarctic ant,Leptothorax acervorum

1. Bergmann's rule states that organisms inhabiting colder environments show an increase in body size or mass in comparison to their conspecifics living in warmer climates. Although originally proposed for homoeothermic vertebrates, this rule was later extended to ectotherms. In social insects, only a few studies have tested this rule and the results were ambiguous. Here, ‘body size’ can be considered at two different levels (the size of the individual workers or the size of the colony). 2. In this study, data from 53 nests collected along altitudinal gradients in the Alps were used to test the hypotheses that the worker body size and colony size of the ant Leptothorax acervorum increase with increasing altitude and therefore follow Bergmann's rule. 3. The results show that the body size of workers but not the colony size increases with altitude. Whether this pattern is driven by starvation resistance or other mechanisms remains to be investigated.     

Variation of chemical composition and antioxidant activity of essential oils of Mentha x rotundifolia (L.) Huds. (Lamiaceae) collected from different bioclimatic areas of Tunisia

The variation of the essential oils composition of 10 Tunisian Mentha x rotundifolia (L.) Huds. Populations and their antioxidant activity were assessed. Essential oils showed high percentages of oxygenated monoterpenes and sesquiterpene hydrocarbons. Rotundifolone, p-menthane-1,2,3-triol, β-caryophyllene and germacrene D were identified as main compounds at the species level. A variation in the essential oil composition was observed according to the populations and ecological factors. The populations 7, 8, 9 and 10 from the upper semi-arid bioclimatic zone and situated at high altitudes, exhibited the highest amount of rotundifolone. The populations 1, 2, 3, 4 and 5 from the lower humid showed a rotundifolone/β-caryophyllene/germacrene D chemotype. The population 6, situated at the lowest altitude, was characterized by the highest amount of p-menthane-1,2,3-triol. The level of antioxidant activity of the populations was linked to their chemical composition difference. The highest scavenging activity and the best ability to reduce ferric ions were recorded for the population 10. The most important capacity to inhibit β-carotene bleaching was revealed for the population 8. For all populations, the antioxidant activities were substantial but lower than antioxidant standards used (Trolox and BHT).The populations (7, 8, 9 and 10) from the upper semi-arid showed the best yields of essential oils and the highest contents of rotundifolone. Chemotypes within these populations could be selected for improvement programs.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

The effect of L-2-amino-4-methoxy-trans-3-butenoic acid on serine hydroxymethyl transferase

The tumour growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the K_m-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation of Walker cells with Ro07-7957 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentration ≤LD₅₀. This increase in enzyme activity does not occur in the presence of cycloheximide. These results suggest that inhibition of serine hydroxymethyltransferase in intact cells is accompanied by an increase in enzyme biosynthesis and that the growth inhibitory property of Ro07-7957 does not involve interference with the conversion of serine to glycine.