Different structures of selected and unselected araB-lacZ fusions

Formation of araB-lacZ coding-sequence fusions is a key adaptive mutation system. Eighty-four independent araB-lacZ fusions were sequenced. All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well-characterized Mu DNA rearrangement process, the strand transfer complex (STC). Five non-standard araB-lacZ fusions isolated after indirect sib selection had novel structures containing back-to-back inverted MuR linkers. The observation that different isolation procedures gave rise to standard and non-standard fusions indicates that cellular physiology can influence late steps in the multi-step biochemical sequence leading to araB-lacZ fusions. Each araB-lacZ fusion contained two novel DNA junctions. The MuR-lacZ junctions showed‘hot-spotting’according to established rules for Mu target selection. The araB-MuR and MuR-MuR junctions all involved exchanges at regions of short sequence homology. More extensive homology between MuR and araB sequences indicates potential STC isomerization into a resolvable four-way structure analogous to a Holliday junction. These results highlight the molecular complexity of araB-lacZ fusion formation, which may be thought of as a multi-step cell biological process rather than a unitary biochemical reaction.     

The roles of starvation and selective substrates in the emergence of araB-lacZ fusion clones.

The araB-lacZ fusion system has been a key case in the 'directed mutation' controversy. Fusions did not occur detectably during normal growth but formed readily after prolonged incubation on selective Ara-Lac medium. To distinguish the roles of starvation stress and selective substrates in coding sequence fusions, we applied sib selection and PCR technologies. Sib selection of the prefusion strain, MCS2, starved under aerobic conditions permitted us to isolate active fusion clones which had never been in contact with arabinose or lactose. Hence, a directive role for selective substrates is not essential. Aerobiosis was necessary for fusions to appear in glucose-starved cultures. The difference in fusion formation between normal and starved conditions is best explained by the response of a signal transduction network to physiological stimuli to activate Mu prophage joining of araB and lacZ sequences. PCR analysis revealed that direct plating on selective Ara-Lac agar yielded mostly a single class of 'standard' fusions, while sib selection yielded a broader spectrum of fusion structures. Standard fusions were found to occur within a narrow 9 bp window in lacZ. The high frequency of standard fusions in glucose-starved cultures suggested efficient and/or specific Mu action.     

Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron.

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.     

Observations on the formation of Chinese desert floras

1. Based upon the analyses in the floristic elements of the three genera (Suaeda, Salsola and Zygophyllum) in different regions we can see that the genesis of our desert floras in these regions is very much diversified. The flora of Songaria is similar to that of the Middle Asia, while the Hosi Corridor seems to be a transitional area very close to Alashan and also related to the Tarim Basin in floristic elements. Thus, we may classify the desert floras into three parts: the flora of Songaria, of Alashan including the Hosi Corridor and of the Tarim Basin including the Tsaidam Basin. The ages and approaches in their formation are different. 2. There are plenty species but no or rare endemics in Songaria. In spring there are a number of ephemeral plants. The variation of aspect is evident. The vegetation cover is abundant. The floristic elements are developed from the flora of Middle Asia and it was formed in Quaternary period. 3. The floristic elements of the Tarim Basin are poor, but there are not few endemics and the distribution of the endemics is much limited. They are of the characteristics of relic species. Therefore it was formed in the Tertiary period and developed in Quaternary period. The elements are related to the Mediterranean flora. 4. There are a large number of endemics and many endemic monotypie genera in Alashan. They represent the flora formed in Tertiary period. Although it is of a special style, it relates both to the Middle-Asian and the Mediterranean flora. 5. The historic causes for the formation of the different floras lie chiefly on: (1) The rise of the Tibetan plateau and mountains strongly changed the climatic and edaphic conditions and in the long course of evolution some species survived or even developed, while the others deteriorated or even died out from the flora. (2) Because the circumstances of transgression or regression of the Tethys were different in these regions. (3) The mountain-making movement, the transgression and regression and the fluence of glaciation, all the mutation of these associated factors modified the climatic zonation and then the plant species changes followed, new species formed and migration of floristie elements occurred. (4) Songaria is the nearest region to the then Sibirian glacier, so the frozen injury to the flora might be the greatest. (5) In the Glacial period the descension of snow line in Songaria was greater than that of the Tarim Basin, so the frozen injurymight be greater.     

Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.     

Observations concerning the sequence of two additional specifically encapsidated RNA fragments originating from the tobacco-mosaic-virus coat-protein cistron.

The incubation of 25-S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial T1 RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the prededing paper, two other species, fragment 41 and fragment 21 are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T1 RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T1 RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, consitute a continuous stretch of 232 nucleotides of the cistron which codes for animo acids 53 to 130 of the coat protein. The order of the fragments in the sequence is 21-41-1. A possible model for the secondary structure of this portion of the sequence is proposed.     

Observations on the formation and persistence of radiopaque transverse lines

The formation and persistence of radiopaque transverse lines have been assessed in the living in clinical studies of growth, nutrition, and disease. Lines have also been used by many researchers in the analysis of patterns of childhood stress in prehistoric skeletal materials, although most of this research has been undertaken on adult bones. However, how reliable and useful are transverse lines for interpretations of stress? The precise etiologies for their appearance are varied and controversial, and lines are also known to resorb during both childhood and the adult years. Sex differences in both their formation and persistence have also been reported. To assess better the viability of lines as indicators of stress, the present research analyzes rates of formation, distribution frequencies, and the persistence of transverse lines in individuals aged birth to 50 + years from a single archaeological population. The results suggest that while transverse lines may be analytically useful as subsidiary criteria for more fully understanding the biological well-being of prehistoric populations, caution should be exercised in the interpretations made concerning childhood stress, particularly when using adult bones.     

Observations on the formation of a pigment by strains of escherichia coli

Summary Escherichia coli cultures grown in salts-synthetic media produce a pigment with tryptophane. Pigment formation is dependent on the tryptophanase activity of the strains and is markedly suppressed by the addition of glucose in the medium. Pigment formation is also highly dependent on light. Other factors influencing this phenomenon are described.