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The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

We have cloned an AP endonuclease gene (APEA) from Dictyostelium discoideum, along with 1.8 kb of the 5' flanking region. There are no introns. The sequence predicts a protein of 361 amino acids, showing high homology to the major human/Escherichia coli exonuclease III family of AP endonucleases. There is 47% identity and 64% similarity to the Ape endonuclease of human cells using the C-terminal 257 amino acids of the Dictyostelium protein. The 104 amino acids on the N-terminus show only low homology with other AP endonucleases. Instead, this region shows high homology with the acid-rich regions of proteins associated with chromatin, such as nucleolins and HMG proteins. The gene is transcriptionally activated up to 7-fold after treatment of cells with sublethal levels of DNA damaging agents, including ultraviolet light, MNNG and bleomycin. Induction does not occur following blocking of replication fork polymerases with aphidicolin. It is not eliminated by treatment with kinase or phosphatase inhibitors. Four DNA damage-sensitive mutants all retained the DNA damage-induced up-regulation.

The repair of cyclobutane pyrimidine dimers was measured under non-replicating conditions in a 2054-bp fragment of a cAMP-inducible cysteine proteinase (CP2) gene in D. discoideum. Overall genomic repair was unaffected by cAMP. The removal of dimers from CP2 in the wild-type NP2 cells as quantified using T4 endonuclease V was independent of transcription and was the same as in the overall genome. In a UV-sensitive radC mutant in which the rate of overall dimer removal was previously shown to be reduced, the initial rate of dimer removal in the uninduced CP2 gene (-cAMP) was 3-fold lower compared to the induced gene (+cAMP), in which repair was identical to that for the induced and uninduced states of NP2. D. discoideum may have two pathways for repairing dimers. One, effective in the wild-type strain but of reduced efficiency in radC repairs dimers equally well independent of transcription and at about the same rate as in the overall genome. A second pathway, retained in radC, repairs dimers more slowly in the overall genome and in the uninduced CP2 gene while undergoing the wild-type rate of repair in the transcriptionally active gene. Hence radC has reduced ability to repair transcriptionally inactive DNA, a defect similar to that of xeroderma pigmentosum group C.

The complete DNA sequence of the 1.7 kilobase pairs (kbp) 5' of the rat growth hormone gene (rGH) has been determined and analyzed for Z-DNA-forming potential. Regions of alternating purine-pyrimidine (APP) sequences located between -1047 and -986 [(GT)31], between -445 and -433 bp, and between -426 and -403 bp relative to the rGH RNA transcription initiation site were identified and shown to form Z-DNA in negatively supercoiled plasmids by two-dimensional gel electrophoresis. Free-energy calculations indicated that Z-DNA forms most readily in the proximal Z-DNA regions. Diethyl pyrocarbonate footprinting of physiologically supercoiled plasmid DNA confirmed the presence of Z-DNA from -444 to -404 bp spanning the two most proximal APP sequences and a short non-APP sequence in between. DNA sequence analysis also predicted a region of DNA curvature near this proximal Z-DNA region. Formation of Z-DNA in the distal Z-DNA region consisting of a (GT)31 repeat was constrained at physiological plasmid superhelical densities. This may be related to the presence of DNA sequences (-1584 to -1559) 512 bp upstream of (GT)31 that undergo cruciform formation and thereby utilize the available free energy. Removal of 580 bp containing the cruciform region resulted in Z-DNA formation within (GT)31, thus demonstrating that deletion mutations can exert topological changes at a distance within the rGH 5'-flanking region. Methylation of two specific cytosines in the rGH 5'-flanking DNA that have been associated with inhibition of rGH promoter activity had no effect on Z-DNA formation. No evidence for DNA secondary structure formation within the rGH second exon-intron or 3'-flanking region was observed. We conclude that the rGH 5'-flanking region undergoes secondary-structure formation at physiological superhelical densities, thus providing a potential mechanism(s) for modulating rGH activity.