Design of ZFRs, Z-sites and test substrates.

<p>(<b>A</b>) Cartoon showing a Tn<i>3</i> resolvase dimer bound to <i>res</i> site I. The red ovals represent the N-terminal (catalytic) and C-terminal (DNA-binding) domains. The motifs recognized by the C-terminal domains are yellow, and the central 12 bp segment (pink) is contacted by the catalytic domains.(<b>B</b>) Cartoon showing a ZFR dimer bound to a Z-site. The zinc finger domains are blue, the motifs they recognize are pale blue, and the ZFR linker peptide is green. Other features are as in <b>A</b>.</p

Casein gene sequences, Z-sites, and recombination assay.

<p>(<b>A</b>) Tn<i>3 res</i> site I (top line) and the seventeen TATA-containing sequences from the β-casein gene intron 1 (Genbank accession number X14711), aligned with the central TATA of site I. Sequences are numbered from the start of exon 1. The motifs in site I bound by the resolvase C-terminal domains are highlighted in yellow. Bases identical to site I within the central 12 bp of the casein sequences are highlighted in pink. The column on the right gives the number of bases identical to the central 12 bp of site I (the number before the slash is for the alignment shown, and the number after the slash is for the sequence aligned in the opposite orientation). The six ‘cas’ sequences, whose central 22 bp sequences were used in the recombination sites analysed in this study, are indicated. The 9 bp sequences flanking the central 22 bp are also shown (highlighted in blue); these sequences would be bound by the ZFR zinc finger domains if the genomic casein gene sequences were to be targeted as Z-sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019537#pone-0019537-g001" target="_blank">Figure 1B</a>). (<b>B</b>) Sequences of site I and Z-sites. The Z-site motifs recognized by the Zif268 domains are highlighted pale blue. The central 22 bp sequences of the ZcasZ sites are from the casein gene sequences shown in part <b>A</b>. Identities of the sequences to the central 12 bp of site I are highlighted pink. Note that the ZcasZ sequences are aligned here to maximize their matches with site I, so some are in the opposite orientation from part A. Motifs recognized by mutant Zif268 domains ZifA and ZifB are highlighted in magenta and cyan respectively. See text for further details. (<b>C</b>) Z-site substrate plasmids and colony colour assay. Recombination between the two Z-sites (boxes) deletes the <i>galK</i> gene, causing colonies to be pale rather than red on MacConkey-galactose indicator plates.</p

Selection of ZFRs to recombine casein gene sequences.

<p>(<b>A</b>) Activity of the progenitor ZFR (ZFR300) on Z-sites with central sequences from Tn<i>3 res</i> site I or the casein gene intron 1. The substrate plasmids each contained two identical Z-sites as indicated above the gel lanes. The numbers below each lane represent the percentage of recombinant plasmids in the recovered DNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019537#s2" target="_blank">Materials and Methods</a>). Images of the ethidium-stained gels are greyscale-inverted, and a uniform background subtraction has been applied. A representative sector of a plate from the corresponding MacConkey-galactose assay is shown above each lane. Annotation: exp, ZFR expression plasmid; nr, non-recombinant substrate plasmid; rec, recombinant (<i>galK</i>⁻) plasmid. (<b>B</b>) Activity of the first round mutant I77L (ZFR310) on the same substrates as in <b>A</b>. (<b>C</b>) Activity of the second round double mutant I77L E132A (ZFR320) on the same substrates as in <b>A</b>.</p

Recombination between non-identical sites.

<p>(<b>A</b>) Substrates containing a Ztn3Z site and a ZcasZ site are recombined by ZFR310. The annotation is as in the previous Figures. The band marked with an asterisk is thought to be single-stranded expression plasmid DNA. (<b>B</b>) Substrates containing a ZcasZ site and Tn<i>3 res</i> site I are recombined when ZFR310 and NM resolvase are co-expressed. (<b>C</b>) A substrate containing an Acas6B site and Tn<i>3 res</i> site I is recombined most efficiently when NM resolvase, ZFR^A320 and ZFR^B320 are co-expressed.</p

Mutant ZFRs selected for recombination of Zcas2Z or Zcas5Z sites.

<p>The Table shows mutants isolated from libraries of mutagenized ZFR300. The left-hand column gives the mutations, and the next column indicates the number of independent isolates. The other columns show the phenotype of each mutant in the MacConkey agar colony colour assay, using substrates with two identical ZcasZ sites as indicated. W, ‘white’ (pale-coloured) colonies; R, red colonies; M, mixtures of pale and red colonies. The letters in bold show the ZcasZ substrate (Zcas2Z and/or Zcas5Z) that was used in the screen from which the mutant was isolated. The four point mutants shown below the thick line were isolated by cloning appropriate fragments from the originally isolated multiple mutant (above the line). n.d., not done.</p

Mutant ZFRs selected for recombination of Zcas6Z sites.

<p>The Table shows the ‘second round’ mutants isolated from screens of ZFR310 mutant libraries (ZFR310 is ZFR300 with the mutation I77L), using a test substrate containing two Zcas6Z sites. The left-hand column gives the mutations, and the next column indicates the number of independent isolates. The other columns show the phenotype of each mutant in the MacConkey agar colony colour assay, using substrates with two identical ZcasZ sites as indicated. W, ‘white’ (pale-coloured) colonies; R, red colonies; M, mixtures of pale and red colonies.</p

Recombination of asymmetric Z-sites.

<p><b>(A)</b> Altered-specificity zinc finger domains target ZFRs to their cognate Z-sites. Substrates containing two identical Z-sites, with either A, B, or Z flanking motifs (Atn3A, Btn3B, Ztn3Z; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019537#pone-0019537-g002" target="_blank">Figure 2B</a>), were recombined by ZFR300 variants with alternative zinc finger domains as indicated immediately above each lane. (<b>B</b>) Efficient recombination at asymmetric (Atn3B) Z-sites requires two ZFR300 variants, with ZifA and ZifB specificity. The letters above each lane indicate the binding domains borne by the expressed ZFRs, with specificity for either Zif268 (Z), ZifA (A), or ZifB (B) motifs. exp(α), pACYC184-based ZFR expression plasmid (pαZFR); exp(β), pBR322-based ZFR expression plasmid (pβZFR). (<b>C</b>) Efficient recombination of AcasB site substrates requires co-expression of ZFR^A320 and ZFR^B320.</p

Comparison regarding general health and behaviour between pups nursed by wild-type dams and pups nursed by α-casein deficient dams.

<p>P-values of parameters without significant differences between group 1 (G1: wild-type pups nursed by wild-type dams) and 2 (G2: wild-type pups nursed by α-casein deficient dams; P-value G1 vs. G2) and group 1 and 3 (G3: heterozygous pups nursed by wild-type dams; P-value G1 vs. G3) at 8 weeks of age (Fisher's exact test and *Mann-Whitney-U test). Constant values indicate that all animals in the groups compared had the same, normal, score.</p

Analysis of milk calcium and phosphate levels and milk protein gene expression.

<p><b>Panel A:</b> Calcium and phosphate content of mouse milk was determined as indicated in the methods section. Concentrations are given in nM. <b>Panel B:</b> Quantitative PCR analysis of α-casein and β-casein gene expression. cDNA derived from representative wild-type, heterozygous [+/−] and homozygous [−/−] α-casein deficient mice was analysed using primer pairs specific for α-casein, β-casein and the reference gene GAPDH. Expression of the casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel C:</b> Quantitative PCR analysis of γ-casein and κ-casein gene expression. Expression of the γ and κ-casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel D:</b> Correlation of casein gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Casein gene expression was correlated with the expression of the reference gene β-actin. Quantification of α-casein was done in 3 [+/+], 7 [+/−] and 5 [−/−] mice. Quantification of β-casein was done in 3 [+/+], 8 [+/−] and 4 [−/−] mice. Quantification of γ- and k-casein was done in 3 [+/+], 3 [+/−] and 3 [−/−] mice. Expression in heterozygous and α-casein deficient mice is presented as percentage of median casein gene expression in wild-type control mice [+/+] (set to 100%). Error bars represent standard deviations. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, p<0.01 by **, and p<0.001 by ***. Exact p values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021775#pone-0021775-t006" target="_blank">table 6</a>.</p

Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.

<p><b>Panel A:</b> Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) <b>Panel B:</b> Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. <b>Panel C:</b> Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.</p