Dieldrin and DDT: effects on sparrow hawk eggshells and reproduction.

Patterns of reproductive failure in declining populations of several European and North American raptorial species were duplicated experimentally with captive American sparrow hawks Falco sparvcrius that were given a diet containing two commonly used organochlorine insecticides. Major effects on reproduction were increased egg disappearance, increased egg destruction by parent birds, and reduced eggshell thickness.

Expression of the Escherichia coli lacZ gene on a plasmid vector in a cyanobacterium.

A biphasic plasmid vector was used to introduce the Escherichia coli K-12 lac operon into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6. The PR-6 transformants expressed beta-galactosidase at nearly as high a level as did Escherichia coli transformants. In order to accomplish this, it was necessary to obtain PR-6 mutants that could be transformed by plasmids with unmodified recognition sites for the endogenous PR-6 restriction endonuclease Aqu I. These mutants were generated by a variation of the ectopic mutagenesis techniques that have been used in other naturally transforming bacteria. The ability to assay the expression of lacZ in PR-6 paves the way for the construction of gene fusions with various PR-6 promoters and quantitation of their expression under specific in vivo conditions.

Specialized transduction with lambda plac5: involvement of the RecE and RecF recombination pathways.

Several aspects of the recombination resulting from lambda plac5 transduction were investigated in strains of Escherichia coli K-12 that use the RecE or RecF recombination pathways. In a RecBC pathway strain, F42lac recombination with lambda plac5 is 20- to 50-fold higher than chromosomal lac times lambda plac5 recombination, and this recombination enhancement is largely dependent on constitutive expression of F42lac fertility functions. Here, it was observed that F42lac fertility functions do not effect the ability of F42lac to recombine with lambda plac5 in a RecE or RecF pathway strain. Therefore, the enhancement observed in a Rec+ (or RecBC pathway) strain is directly dependent on the recBC gene product. The end product of recombination between lambda plac5 and either F42lac or chromosomal lac in RecE and RecF pathway strains was monitored by scoring for addition and substitution transductants. It was observed that the percentage of addition transductants was lower in all cases for RecE and RecF pathway strains as compared with RecBC pathway or a recB strain. It is concluded that the introduction of sbcA or sbcB into a recB strain produces a change in recombination mechanism that is reflected in the nature of the end product of recombination.

Genes from the cyanobacterium Agmenellum quadruplicatum isolated by complementation: characterization and production of merodiploids.

The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described. Although our search for such genes has not been exhaustive, it appears that complementation of E. coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes. Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons. We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained. Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector. Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium.

Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli.

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV- and temperature-sensitive. The defects of both ssbH55Y (ssb-1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild-type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild-type SSB protein.

Mutations in the bacteriophage lambda pL/oL region that spontaneously occur in plasmid pRPZ126.

In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups. The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage lambda immunity region with the cI857 allele so that plasmid-containing cells shifted to 42 degrees C survive only if the expression of the lambda kil gene is prevented by mutation. 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size. The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever. In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the lambda pL/oL region of the mutant plasmid. Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair deletion in the pL/oL region of the plasmid. The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region. The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids. 8 out of 9 of these transition mutations occurred next to the 3' end of 3 different 5'-PyGGNPuNTG-3' sequences in the lambda operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986). The 9th mutation, where the A-T to G-C transition occurred one basepair away from the lambda operator, was adjacent to a very similar sequence.

Characterization of the types of mutational events that spontaneously occur in a plasmid system.

The immunity region from a cI857 derivative of bacteriophage lambda has been cloned into the EcoRI site of pBR322 to produce a plasmid that can be used to analyze spontaneous mutagenesis. Cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the lambda fragment at non-permissive temperature. 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different recA- E. coli strains have been collected, and the nature of the plasmid mutations obtained has been analyzed. All of the subcultures contained mutants that allowed growth at the restrictive temperature without showing a detectable change in plasmid size. 75% of the total mutants fell in this class. More than half of these mutations involved the lambda leftward promoter, pL, and such mutants were found in all 20 subcultures. The remaining 25% of the mutations involved a change in plasmid size and mutations of this class were found in 18 of 20 subcultures. 12% of the total mutants (found in 16 of 20 subcultures) had an insertion of IS1 in the region between pL and the lambda kil gene. 6% of the total mutants had undergone an IS1-mediated deletion, while 1% were mixed colonies in which multiple IS1-mediated events had occurred. About 1% of the total mutants had undergone complex IS1-mediated DNA rearrangement(s) that have not yet been characterized. In total, 11 of 20 subcultures yielded isolates where IS1-mediated rearrangements had occurred. The remaining 4% of the mutations included insertions of IS5, IS30, and an IS1 family member that appears to be IS1T as well as IS1T-mediated deletions and deletions that do not appear to have been mediated by any insertion sequence. A mutant with both an IS1 insertion and an alteration involving pL has also been isolated.

Armed services vocational aptitude battery predictors of entry-level radiography students' success.

This study assessed the Armed Services Vocational Aptitude Battery (ASVAB) predictors of student success in the diagnostic imaging course at the Air Force's School of Health Care Sciences. Regression analyses provided two significant predictors (p < 0.05) of student success as identified by final grade. Those predictors were the mechanical and electronics scores on the ASVAB. Also, both scores were significantly different (p < 0.05) among those who passed, washed back (passed but required excess time), and disenrolled. Because a significant number of individuals take the ASVAB, entry-level radiography programs should consider using these predictors for admissions decisions.

Predictors of students' success in the Medical Service Apprentice course.

This study assessed the predictors of students' success in the 383d Training Squadron's Medical Service Apprentice course at Sheppard Air Force Base, Texas. Correlation and regression analyses provided seven predictors of students' success. These predictors were the Armed Services Vocational Aptitude Battery Mechanical score, block 1 grade, block 3 grade, block 3 student hours, block 4 grade, block 4 student hours, and National Registry Emergency Medical Technician Test 1 grade. From these predictors, researchers ascertained demarcation points to provide direction for instructors and students. Using this guidance, proactive intervention by instructors and the Student Learning Center will improve the success of Medical Service Apprentice course students. Further research conducted by the 882d Training Group to evaluate predictors of success will yield a seamless evaluation process. This process will establish an improved proactive educational system enhancing the career field selection process and the quality of student education.

Predictors of students' success in the Air Force School of Health Care Sciences.

This study assessed the predictors for students' success in the 882d Training Group's (Air Force School of Health Care Sciences) allied health courses at Sheppard Air Force Base, Texas. Correlation and regression analyses provided many predictors of students' success. From these predictors, the researchers ascertained demarcation points to provide direction for instructors and students. Using these parameters, proactive intervention by instructors, students, and the Student Learning Center will improve the success of allied health students. Further research conducted by the 882d Training Group in evaluating predictors of success will yield a seamless evaluation process. This process will establish an improved proactive educational system enhancing the career field selection process and the quality of students' education, as well as decrease costs.

Air Force School of Health Care Science's quality of life study.

This was an empirically based assessment of non-prior service students' quality of life in the Air Force's School of Health Care Sciences. Analysis provided five results: (1) The overall quality of life at the school was good. (2) The variables accounting for student unhappiness were dormitory unsuitability and the students not being in their top-three career choices. (3) Structural changes were required at the dormitories. (4) The desire to succeed and how to achieve that success were the most important interests for students. (5) Loved ones and student independence were the greatest indicators of motivation. The findings resulted in three immediate corrections and two long-term recommendations to improve students' quality of life. The two long-term recommendations were to have an educational psychologist intervene when students are having significant learning problems, and to alter the selection process for recruiting. Both immediate corrections and long-term recommendations are useful for sister services.

Use of the Escherichia coli SSB gene to prevent bioreactor takeover by plasmidless cells.

Reactor takeover by plasmidless cells is a major problem encountered when producing proteins from plasmid-borne genes in genetically engineered bacteria. We have approached this problem by deleting the essential ssb gene from the Escherichia coli chromosome and placing it on a plasmid. Plasmidless cells do not accumulate even after growing such strains under non-selective continuous culture conditions for extended periods of time. Other ssb-containing plasmids can be readily introduced into this E. coli strain by a plasmid-displacement technique. Using this system, we have achieved very high levels of beta-lactamase production in continuous culture without selective pressure.

Transformation in cyanobacteria.

The lack of any known transduction or indigenous conjugation systems has left transformation as the major means for genetic manipulations in cyanobacteria. Studies of transformation in cyanobacteria generally have dealt with one of two distinct areas. The first area is genomic transformation where internalized donor DNA recombines with chromosomally located genes. Chromosomal transformation can be a powerful tool for genetic mapping and mutagenesis. The second area is plasmid transformation where internalized plasmid donor DNA becomes established as an independent replicon in the recipient cyanobacterium. This second area has received a great deal of attention because it allows the generation of merodiploids for studies of genetic regulation and control and because it potentially allows the expression of foreign genes in an oxygenic photoautotroph. This article will attempt to describe the development of our current understanding of these two types of genetic transformation in cyanobacteria.

Recombination properties of P1 dlac.

The P1 dlac prophage plasmid of Escherichia coli K-12 has been utilized as the recipient DNA substrate in experiments with lambda plac5 transduction and with Hfr and F' conjugation. The P1 dlac plasmid does not recombine with lambda plac5 at the elevated levels seen for the F42lac plasmid. Recombination between lambda plac5 and P1 dlac is essentially indistinguishable from recombination between lambda plac5 and a chromosomal lac gene in tems of both level of recombination and recombination pathway (RecBC, RecE, and RecF) dependence. The initiation of recombination between P1 dlac and lac genes from an Hfr or F' donor is severalfold more efficient than it is for a recipient chromosomal lac gene.

Enhanced recombination between F42lac and lambda plac5: dependence on F42lac fertility functions.

F42lac recombination with lambda plac5 is normally twentyfold to fiftyfold higher than recombination between lambda plac5 and a chromosomal lac gene. The presence of an fi+ R1 plasmid in the same cell as F42lac dramatically reduces this enhanced recombination level while the fi- R1drd19 plasmid has little effect. When F42lac traJ90 is tested in a sup+ strain, it shows a sharp reduction in recombination with lambda plac5 that can be largely reversed by the presence of a supF mutation that partially suppresses the traJ90 nonsense mutation. It is concluded that the enhanced recombination between F42lac and lambda plac5 is largely dependent on the constitutive expression of F42lac fertility functions.

Transfection in pneumococcus: single-strand intermediates in the formation of infective centers.

Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection.

General recombination in Escherichia coli K-12: in vivo role of RecBC enzyme.

Heterozygous lacZ- merodiploids of Escherichia coli K-12 have been used to study the role of the RecBC enzyme in general recombination. The transcribable intermediate assay detects the product of early steps in recombination without requiring the formation of viable recombinant colonies. Recombination is initiated by infection with lambda precA+. We have found that transcribable intermediate formation in crosses between F42 lac and chromosomal lac is dependent on F fertility functions and an active RecBC enzyme. Thus, the products of the recB and recC genes are required in early steps of recombination between these two substrates. Introduction of the F42 lac donor DNA by conjugation immediately after infection with lambda precA+ abolishes the requirement for an active RecBC enzyme.

Heterospecific transformation among cyanobacteria.

Heterospecific transformation occurred between cyanobacteria currently classified in either the genus Synechococcus or Synechocystis. Cyanobacterial strains 73109 and 6906 were capable of physiological transformation.