Do soil cadmium concentrations decline after phosphate fertiliser application is stopped: A comparison of long-term pasture trials in New Zealand?

Decreasing soil cadmium (Cd) is one method of removing Cd from the food chain. Phosphorus (P) fertilisers are a major source of Cd inputs into soil. Stopping P fertiliser should theoretically decrease Cd inputs and soil Cd accumulation, but there are few field data to show if this occurs. We examined three long-term grazed pasture trials in New Zealand (Ballantrae, Winchmore and Whatawhata) where P fertiliser had been applied (from 10 to 100 kg P ha-1 yr-1) for up to six years and then stopped for 10 to 26 years. Stopping P fertiliser applications reduced soil Cd concentrations at Winchmore and Whatawhata where P had been applied at ≥34 kg P ha-1 yr-1. No reductions occurred below this rate nor at Ballantrae where only 10 years post P-application data were available. Decreases were ascribed to moderate rainfall (1630 mm at Whatawhata and 740 mm rainfall plus 770 mm irrigation at Winchmore) that enhanced Cd leaching and may have been aided at Winchmore by a decrease in soil pH over time (0.4 units). However, because stopping P fertiliser inputs may quickly impair pasture production, additional strategies may be required to decrease soil Cd quickly.

Seventy years of data from the world's longest grazed and irrigated pasture trials.

Pastures are the most widespread land use, globally. The Winchmore trials were established in 1948-1949 in Canterbury, New Zealand and examined either different rates of phosphorus (P) fertiliser on the same irrigation schedule (Fertiliser trial), or different irrigation scheduling at the same rate of P application (Irrigation trial). About 96,000 records of soil chemistry and physical data and pasture yield and botanical composition are available along with nearly 7000 soil samples. These data have been used in 475 publications that have explored topics as diverse as: improvements in sheep, dairy and deer production; the efficacy and scheduling of irrigation; improvements in pasture and crop production; agronomic and environmental soil and water research; and entomology. In addition to above topics, these data are invaluable for calibrating models to predict long-term issues like the accumulation of soil carbon or contaminants like cadmium and informing policy on climate change and agricultural practices. The data and soil samples are available for use and may yet yield discoveries, unforeseen 70 years ago.

Cadmium uptake by onions, lettuce and spinach in New Zealand: Implications for management to meet regulatory limits.

Paired soil and plant samples collected from the main commercial growing areas for onions (Allium cepa), lettuce (Lactuca sativa) and spinach (Spinacia olearacea) in New Zealand were used to assess the influence of plant and soil factors on cadmium (Cd) uptake in these crops. Differences in Cd concentration between eight lettuce sub-types were not consistent across sites, nor were differences in Cd concentrations in three crisphead cultivars assessed at two sites. Similarly, differences in Cd concentrations between four onion cultivars were inconsistent across sites. Mean lettuce Cd concentrations in eight lettuce varieties (range 0.005-0.034 mg∙kg-1 (fresh weight, FW) were markedly lower than those in baby leaf and bunching spinach, (range 0.005-0.19 mg∙kg-1 FW). Significant regional variation was observed in Cd concentrations in one onion cultivar (mean range 0.007-0.05 mg∙kg-1 FW). Soil Cd concentration, pH and region were statistically significant predictors of onion Cd concentration, explaining low (38% for soil Cd and pH) to moderate (50% for all three parameters) percentage of the variation. Soil Cd concentration and exchangeable magnesium or total carbon were statistically significant predictors of Cd concentration in baby leaf and bunching spinach, respectively, explaining a moderate percentage (49% and 42%) of the variation in Cd concentration. Increasing pH and soil carbon may assist in minimising Cd uptake in onion and bunching spinach, respectively. The low to moderate proportion of explained variation is partly attributable to the narrow range in some measured soil properties and indicates factors other than those assessed are influencing plant uptake. This highlights a challenge in using these relationships to develop risk-based soil guideline values to support compliance with food standards. Similarly, the inconsistency in Cd concentrations in different cultivars across sites highlights the need for multi-site assessments to confirm the low Cd accumulation status of different cultivars.

An assessment of the effect of contact time on cadmium phytoavailability in a pasture soil.

The length of time cadmium (Cd) is in contact with the soil has been recognised as a factor affecting phytoavailability, but the extent of this process is currently poorly understood. This study used isotopic dilution techniques (E and L values) to determine the effect of contact time on Cd phytoavailability from soil collected from a long-term phosphorus (P) fertiliser trial. Cadmium phytoavailability was determined in soil that was last fertilised with soluble Cd from P fertiliser 17 years prior to sampling (residual plots) and soil that received annual applications of P fertiliser until sampling (continuous plots). It was found that both E values and L values increased with P fertiliser (viz Cd) inputs and were significantly related to each other (r 2 = 0.82 P < 0.005). There was however no significant difference (P < 0.05) in the percentage of total Cd that was phytoavailable calculated using E values (E%) between the continuous (mean 51 %) and the residual plots (mean 51 %). There was also no significant difference (P < 0.05) in the percentage of total soil Cd that was phytoavailable calculated using L values (L%) between the continuous (mean 77 %) and residual plots (mean 87 %). These results suggest that despite Cd being in contact with the soil for 17 years, there was no difference in the size of the phytoavailable Cd pool compared to recent Cd inputs. This study should be repeated for other soil types and factored into any analysis for the long-term implications of ongoing Cd accumulation in soil on future landuse.

Quality defects in market beef and dairy cows and bulls sold through livestock auction markets in the Western United States: II. Relative effects on selling price.

Relative effects of Beef Quality Assurance (BQA)-related defects in market beef and dairy cows and bulls on selling price at auction was evaluated during 2008. The presence and severity of 23 BQA-related traits were determined during sales in Idaho, California, and Utah. Overall, 18,949 unique lots consisting of 23,479 animals were assessed during 125 dairy sales and 79 beef sales. Mean sale price ± SD (per 45.5 kg) for market beef cows, beef bulls, dairy cows, and dairy bulls was $45.15 ± 9.42, $56.30 ± 9.21, $42.23 ± 12.26, and $55.10 ± 9.07, respectively. When combined, all recorded traits explained 36% of the variation in selling price in beef cows, 35% in beef bulls, 61% in dairy cows, and 56% in dairy bulls. Premiums and discounts were determined in comparison with a "par" or "base" animal. Compared with a base BCS 5 beef cow (on a 9-point beef scale), BCS 1 to 4 cows were discounted (P < 0.0001), whereas premiums (P < 0.05) were estimated for BCS 6 to 8. Compared with a base BCS 3.0 dairy cow (on a 5-point dairy scale), more body condition resulted in a premium (P ≤ 0.001), whereas a less-than-desirable BCS of 2.0 or 2.5 was discounted (P < 0.0001). Emaciated or near-emaciated cows (beef BCS 1 or 2; dairy BCS 1.0 or 1.5) were discounted (P < 0.0001). Compared with base cows weighing 545 to 635 kg, lighter BW beef cows were discounted (P < 0.0001), whereas heavier beef cows received (P < 0.05) a premium. Compared with a base dairy cow weighing 636 to 727 kg, lighter BW cows were discounted (P < 0.0001), whereas heavier cows (727 to 909 kg) received a premium (P < 0.01). Beef and dairy cows with any evidence of lameness were discounted (P < 0.0001). Presence of ocular neoplasia in the precancerous stage discounted (P = 0.05) beef cows and discounted (P < 0.01) dairy cows, whereas at the cancerous stage, it discounted (P < 0.0001) all cows. Hide color influenced (P < 0.0001) selling price in beef cattle but had no effect (P = 0.17) in dairy cows. Animals that were visibly sick were discounted (P < 0.0001). Results suggest that improving BCS and BW, which producers can do at the farm or ranch level, positively affects sale price. Furthermore, animals that are visibly sick or have a defect associated with a possible antibiotic risk will be discounted. Ultimately, animals with minor quality defects should be sold in a timely manner before the defect advances and the discount increases.

Survey of quality defects in market beef and dairy cows and bulls sold through livestock auction markets in the Western United States: I. Incidence rates.

A survey was conducted to quantify incidence of Beef Quality Assurance (BQA)-related defects in market beef and dairy cows and bulls selling at auction during 2 seasons in 2008. Twenty-three BQA-related traits were evaluated by 9 trained personnel during sales at 10 livestock auction markets in Idaho (n = 5; beef and dairy), California, (n = 4; dairy only), and Utah (n = 1; beef and dairy). Overall, 18,949 unique lots (8,213 beef cows, 1,036 beef bulls, 9,177 dairy cows, and 523 dairy bulls,) consisting of 23,479 animals (9,299 beef cows, 1,091 beef bulls, 12,429 dairy cows, and 660 dairy bulls) were evaluated during 125 sales (64 spring, 61 fall) for dairy and 79 sales (40 spring, 39 fall) for beef. The majority of market beef cows and bulls (60.9 and 71.3%, respectively) were predominantly black-hided, and the Holstein hide pattern was observed in 95.4 and 93.6% of market dairy cows and bulls, respectively. Market cattle weighed 548 ± 103.6 kg (beef cows), 751 ± 176.1 kg (beef bulls), 658 ± 129.7 kg (dairy cows), and 731 ± 150.8 kg (dairy bulls). Most beef cows (79.6%) weighed 455 to 726 kg, and most beef bulls (73.8%) weighed 545 to 954 kg, respectively. Among market beef cattle, 16.0% of cows and 14.5% of bulls weighed less than 455 and 545 kg, respectively, and 63.7% of dairy cows and 81.5% of dairy bulls weighed 545 to 817 kg or 545 to 954 kg, respectively. However, 19.5% of dairy cows and 13.1% of dairy bulls weighed less than 545 kg. Mean BCS for beef cattle (9-point scale) was 4.7 ± 1.2 (cows) and 5.3 ± 0.9 (bulls), and for dairy cattle (5-point scale) was 2.6 ± 0.8 (cows) and 2.9 ± 0.6 (bulls). Some 16.5% of beef cows and 4.1% of beef bulls had a BCS of 1 to 3, whereas 34.8% of dairy cows and 10.4% of dairy bulls had a BCS of 2 or less. Emaciation (beef BCS = 1, dairy BCS = 1.0) or near-emaciation (beef BCS = 2, dairy BCS = 1.5) was observed in 13.3% of dairy cows and 3.9% of beef cows. Among beef cattle, 15.1% of cows and 15.4% of bulls were considered lame. In contrast, 44.7% of dairy cows and 26.1% of dairy bulls were lame. Ocular neoplasia (cancer eye) was observed in only 0.6% of beef cows, 0.3% of beef bulls, 0.3% of dairy cows, and 0.0% of dairy bulls. However, among animals with ocular neoplasia, it was cancerous in 34.4% of beef bulls, 48.0% of dairy cows, and 73.3% of beef cows. In conclusion, numerous quality defects are present in market beef and dairy cattle selling at auction in the Western United States, which could influence their value at auction.

The mitogen-activated/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 induces glial fibrillary acidic protein expression and reduces the proliferation and migration of C6 glioma cells.

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.

Field evaluation of Cd and Zn phytoextraction potential by the hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri.

Field trials were undertaken to investigate the effect of the application of metal mobilizing agents, different sowing strategies and length of growing season on the extraction of Cd and Zn from soils by Thlaspi caerulescens and Arabidopsis halleri. None of the mobilizing agents used enhanced metal accumulation by T. caerulescens. Between 1998 and 2000, on average across plots where Cd or Zn exceeded allowable limits, T. caerulescens removed 1.3 and 0.3% of the total soil Cd and Zn. In one season when T. caerulescens was grown for 14 months, 21.7 and 4.4% of the total soil Cd and Zn was removed. This was larger than values found when T. caerulescens was grown for 4 months. A. halleri accumulated similar concentrations of Zn, but lower Cd concentrations than T. caerulescens. The results indicate that metal phytoextraction using T. caerulescens can be used to clean up soils moderately contaminated by Cd.

Field evaluation of in situ remediation of a heavy metal contaminated soil using lime and red-mud.

We evaluated the effectiveness of lime and red mud (by-product of aluminium manufacturing) to reduce metal availability to Festuca rubra and to allow re-vegetation on a highly contaminated brown-field site. Application of both lime and red mud (at 3 or 5%) increased soil pH and decreased metal availability. Festuca rubra failed to establish in the control plots, but grew to a near complete vegetative cover on the amended plots. The most effective treatment in decreasing grass metal concentrations in the first year was 5% red mud, but by year two all amendments were equally effective. In an additional pot experiment, P application in combination with red mud or lime decreased the Pb concentration, but not total uptake of Pb in Festuca rubra compared to red mud alone. The results show that both red mud and lime can be used to remediate a heavily contaminated acid soil to allow re-vegetation.

The over-expression of the wild type or mutant forms of the presenilin-1 protein alters glycoprotein processing in a human neuroblastoma cell line.

Mutations in the presenilin proteins (PS1 and PS2) are responsible for more than 70% of the cases of the familial form of Alzheimer's disease (FAD). The proteins are expressed in the cell at a low level, primarily in the endoplasmic reticulum and cis Golgi, where they have been proposed to play a role in protein processing. As protein glycosylation is a key post-translational event that occurs within the Golgi, we have investigated the effect of altered PS1 expression levels on the protein glycosylation pattern using the SH-SY5Y human neuroblastoma cell line. In cells over-expressing either the wild type or mutant (M146L) PS1-FAD proteins, there was a decrease in the expression levels of protein-bound alpha2,3-linked sialic acid residues at the level of the cell membrane. This was particularly manifest as a significant decrease in the expression of the polysialic acid chain that is linked to the core oligosaccharide of the neural cell adhesion molecule in an alpha2,3 bond. These results suggest that the over-expression of either the wild type or mutant PS1 disturbs glycoprotein processing within the Golgi.

SELEX selection of high-affinity oligonucleotides for bacteriophage Ff gene 5 protein.

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA.

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.

Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS.

TREK-1 is a member of the two-pore-domain potassium channel family which is expressed predominantly in the CNS. Using an anti-peptide polyclonal antiserum, we have determined the distribution of TREK-1 in the brain and spinal cord of adult rats. Specificity of the antiserum was tested using a TREK-1-transfected cell line and confirmed with c-myc-tagged TREK-1. In thin tissue sections, immunoreactivity was widespread throughout the rat brain and spinal cord. TREK-1-like signals were observed in the cerebral cortex, basal ganglia, hippocampus, and various other subcortical nuclei in the hypothalamus, thalamus, mesencephalon and rhombencephalon. TREK-1 labelling appeared to be over the entire cell membrane, including the cell body and processes. Cells that morphologically resembled projection neurones and interneurones but not glial cells were labelled. As interneurones and known GABAergic projection neurones were the predominant population labelled, we investigated the possibility that TREK-1 is expressed in GABA-containing neurones using a specific anti-GABA antiserum. Expression of TREK-1 in GABA-containing neurones was observed in a number of areas, including the isocortex, hippocampus and thalamus. Thus, TREK-1 expression defines a unique and specific subset of interneurones and principal cells. These studies indicate a widespread distribution of TREK-1 potassium channels throughout the rat brain and spinal cord, with expression in a number of areas being demonstrated to be present on GABA-containing neurones.

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

Expression, purification, and functional analysis of the human serine protease HtrA2.

HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.

Development of Neoepitope Antibodies Against the ß-Secretase Cleavage Site in the Amyloid Precursor Protein.

A detailed understanding of the biochemical events leading to the proteolytic excision of the ß-amyloid peptide (A ß) from the amyloid precursor protein (APP) has eluded many researchers. This is largely because the measurement of the various APP processing products is technically challenging owing to their low levels of production in in vitro and in vivo test systems. Sequence analysis of products in cell cultures, cerebrospinal fluid (CSF), and amyloid plaques has been used to predict the major cleavage sites resulting from the ß- and γ-secretase proteolytic activities that release the Aß peptide from APP (1 -3). More routine identification of the secretase activities has relied on the specificity and sensitivity of antibodies raised to the predicted cleavage products and has been impeded by the difficulties associated with the generation of such reagents.

Alzheimer's disease: correlation of the suppression of beta-amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate.

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.

Circular dichroism and electron microscopy of a core Y61F mutant of the F1 gene 5 single-stranded DNA-binding protein and theoretical analysis of CD spectra of four Tyr --> Phe substitutions.

A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotide binding modes for Y61F and wild-type g5p. There are n = 4 and n congruent with 2.5 modes for binding to poly[d(A)] at low ionic strengths, but n = 4, n = 3, and n congruent with 2-2.5 modes for binding to fd single-stranded viral DNA (fd ssDNA), where n is the number of nucleotides occluded by each bound g5p monomer in a given mode. Y61F g5p has slightly reduced affinity in the n = 4 mode. Electron microscopy showed that Y61F g5p forms left-handed nucleoprotein superhelices indistinguishable from wild-type. Progression from binding to fd ssDNA in the n = 4 to n = 3 to n congruent with 2-2.5 mode is accompanied by an increase in the number of helical turns, an increase from (7.7 +/- 0.3) to (9.5 +/- 0.3) to ( approximately 10-13) g5p dimers per turn, and a decrease in the number of DNA nucleotides per turn. From CD spectra for four of five possible Y --> F g5p mutants, we infer that the fifth tyrosine, Tyr 56, contributes strongly to the CD. Retention of a strong 229 nm CD band in all mutants indicates that all retain elements of the native structure. Spectra of Y26F, Y34F, and Y61F g5p imply limited mobility of the replacement Phe. Comparison of measured with calculated CD spectra also suggests limited mobility for Tyr 26 and Tyr 34 in g5p in solution, and provides new information that the g5p structure in solution may be dominated by Tyr 41 rotamers differing from that stabilized in the crystal.