Crop Rotations and Poultry Litter Affect Dynamic Soil Chemical Properties and Soil Biota Long Term.

Dynamic soil chemical interactions with conservation agricultural practices and soil biota are largely unknown. Therefore, this study aims to quantify long-term (12-yr) impacts of cover crops, poultry litter, crop rotations, no-tillage, and their interactions on dynamic soil properties and to determine their relationships with nutrient cycling, crop yield, and soil biodiversity (soil microbial and earthworm communities). Main effects were 13 different cropping sequences of soybean [ (L.) Merr.], corn ( L.), and cotton ( L.) at the Research and Education Center at Milan, TN, and eight sequences of corn and soybean at the Middle Tennessee Research and Education Center, Spring Hill, TN. Sequences were repeated in 4-yr phases from 2002 to 2014. Split-block cover crop treatments consisted of winter wheat ( L.), hairy vetch ( Roth), poultry litter, and a fallow control. Soil C and nutrient fluxes were calculated at surface (0-5 cm) and subsurface (5-15 cm) layers during Years 0, 2, 4, 8, and 12. After 12 yr, weighted means (0-15 cm) of soil pH, P, K, Ca, Mg, total N, and C were greater under poultry litter-amended soils compared with cover crops ( < 0.05). In addition, continuous corn sequences resulted in greater soil K, N, and C concentrations than soybean-soybean-corn-corn rotations ( < 0.05). Poultry litter treatments were positively correlated with greater soil fertility levels, as well as higher crop yield and soil biodiversity. These results underscore linkages between manure additions and cropping sequences, within the nutrient cycling, soil health, and crop production continuum.

Modulation of gene expression in the embryonic digestive tract of C. elegans.

The Caenorhabditis elegans digestive tract is composed of four distinct modules derived from separate cell lineages: anterior pharynx from the ABa lineage, posterior pharynx from the MS lineage, gut from the E lineage, and rectum from the ABp lineage. The C. elegans gut esterase gene (ges-1) is normally expressed in the embryonic gut or E lineage. However, expression ges-1 can be switched into cells of the embryonic pharynx and tail by virtue of deleting a tandem pair of WGATAR sites in the ges-1 promoter. Here, we use both laser ablation experiments and genetic analysis to show that cells expressing the WGATAR-deleted ges-1 transgene belong to all three nongut lineages of the digestive tract: ABa, MS, and ABp. We also show that the molecular size and spatial distribution of ges-1 mRNA transcripts produced by either the WGATAR-deleted ges-1 transgene or the undeleted ges-1 control transgene appear correctly regulated, suggesting that the spatial switch in ges-1 expression occurs at the level of transcription initiation. We further show that both the WGATAR-deleted and the undeleted ges-1 transgenes respond appropriately to mutations in a series of maternal effect genes (skn-1, mex-1, pie-1, and pop-1) that alter early blastomere fate. Moreover, the pharynx/tail expression of the WGATAR-deleted ges-1 transgene is abolished by mutations in the zygotic gene pha-4. Finally, we use imprecise transposon excision to produce two independent C. elegans strains with 1- to 2-kb deletions that remove the tandem WGATAR sites from the promoter of the endogenous chromosomal ges-1 gene: in both of these strains, ges-1 is not expressed in the embryonic gut but is expressed in cells of the embryonic pharynx; pharynx expression is weak but incontrovertible. Overall, our results validate previous transgenic analysis of ges-1 control and show further that ges-1 appears to be regulated in a system-specific, rather than a lineage-specific, manner. The multiple facets of ges-1 expression provide an opportunity to investigate how a multicomponent organ system such as the digestive tract is established from diverse cell lineages.

A gut-to-pharynx/tail switch in embryonic expression of the Caenorhabditis elegans ges-1 gene centers on two GATA sequences.

The Caenorhabditis elegans ges-1 gene (gut esterase No. 1) is expressed only in the intestinal lineage, beginning when the developing gut has only four to eight cells. We analyze the sequence requirements for this tissue-specific gene regulation by injecting deleted/mutated constructs of the ges-1 gene into a viable ges-1 (null) strain of worms and assaying heritably transformed embryos by esterase histochemistry. Many deletion constructs accurately reconstitute the wildtype gut-specific ges-1 expression. However, deletions in the neighborhood of 1100 bp upstream of the ges-1 ATG abolish ges-1 expression in the developing gut, while at the same time activating ges-1 expression in cells of the pharynx/tail that appear to belong to the sister lineage of the gut. Deletions of a 36-bp DNA region containing two tandem WGATAR sequences are sufficient to cause this gut-to-pharynx/tail switch in expression pattern. Deletion of either one of the WGATAR sites or deletion of an adjoining downstream region directs ges-1 expression only in a restricted set of cells of the anterior gut. The ges-1 GATA region acts like a gut-specific enhancer in that: (i) it restores ges-1 gut expression when reinserted elsewhere into the GATA-deleted ges-1 gene; and (ii) multiple copies direct gut expression of an hsp16-lacZ reporter gene. The ges-1 GATA-region also acts as the site of the pharynx/tail repression in that reinsertion elsewhere into the GATA-deleted ges-1 construct causes repression of ges-1 in the pharynx/tail. However, multiple copies of the GATA region are not able to repress the heat-induced expression of an hsp16-lacZ reporter gene, suggesting that the pharynx/tail repression mechanism is specific to the ges-1 environment. Finally, mutation rather than deletion of the individual GATA sequences suggests that gut activation and pharynx/tail repression may be due to separate factors. We present a molecular model that summarizes these results. The ges-1 control circuitry appears surprisingly complex for what might have been expected to be the simplest possible example of a nonessential gene expressed early in a clonal embryonic lineage.

The gut esterase gene (ges-1) from the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

The ges-1 gene codes for a non-specific carboxylesterase that is normally expressed only in the intestine of the nematode Caenorhabditis elegans. In the current paper, we describe the cloning and characterization of the ges-1 gene from C. elegans, as well as the homologous gene from the nematode Caenorhabditis briggsae. The ges-1 esterases from the two nematodes are 83% identical at the amino acid level and contain regions of significant similarity to insect and mammalian esterases; these conserved regions can be identified with residues believed to be necessary for esterase function. The ges-1 mRNAs from both C. elegans and C. briggsae are trans-spliced. The coding regions, the codon bias and the splicing signals of the two ges-1 genes are quite similar and most (6/7) of the intron positions are retained precisely. Yet, the flanking sequences of the two ges-1 genes appear to have diverged almost completely. For example, the C. elegans ges-1 5'-flanking region (as well as several introns) contains copies of three different SINE-like sequences, previously identified near the hsp-16 genes, near the unc-22 gene and in a repetitive element CeRep-3; none of these elements are found in the C. briggsae ges-1 gene. We show that: (1) the C. elegans ges-1 gene can be used to transform C. briggsae, whereupon expression of the exogenous ges-1 gene is confined to the C. briggsae intestine; (2) the ges-1 homologue cloned from C. briggsae can be transformed into C. elegans, whereupon it is expressed largely in the C. elegans intestine; and (3) a 5'-deletion of the C. elegans ges-1 gene that we have previously shown to be expressed in the C. elegans pharynx is also expressed in the pharynx of C. briggsae (either in the presence or absence of vector sequences). These results suggest that the ges-1 gene control circuits have been maintained between the two nematode species, despite the divergent 5'-flanking sequences of the gene. This raises the question of the evolutionary distance between C. elegans and C. briggsae and we attempt to estimate the C. elegans-C. briggsae divergence time by analysing the rate of synonymous substitutions in coding regions of ges-1 and six other C. elegans-C. briggsae gene pairs. We propose a new method of analysis, which attempts to remove rate differences found between different genes by extrapolating to zero codon bias.(ABSTRACT TRUNCATED AT 400 WORDS)

An M13 vector library for cloning DNA with four nucleotide 3' overhangs.

A flexible M13 vector library incorporating the BstXI site has been developed. DNA cut by any currently commercially available restriction endonuclease that generates a 4-nucleotide (nt) 3' overhang can be ligated into a specific clone of the library. The BstXI enzyme recognizes a 6-bp bipartite palindromic sequence. The central nucleotides are not specified, and form a 4-base, 3' overhang when cut by BstXI. 5' CCANNNNN NTGG GGTN NNNNNACC 5' Since the 4-base overhang formed is not part of the BstXI recognition sequence, it is possible to generate a library of 256 different clones by introducing the BstXI site, 151 of the possible 256-member library have been isolated, including all 13 M13BF clones in which the overhang formed by BstXI digestion is complementary to those formed by currently available restriction endonucleases. Of these 13 vectors, BstXI digestion of six clones results in nonpalindromic cohesive ends and should facilitate in vitro tandem gene amplification. The BstXI site is adjacent to the four codons corresponding to the factor Xa recognition sequence. Hence the vector library could facilitate the expression of a fusion protein that could be proteolytically cleaved by factor Xa.

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli.

LacI- mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G:C----C:G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G:C----C:G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after the two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication and repair mechanisms.

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein.

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

DNA sequence analysis of mutagenicity and site specificity of ethyl methanesulfonate in Uvr+ and UvrB- strains of Escherichia coli.

EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strains; G:C----A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed premutagenic lesion, O6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.