Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

Fine Structure Mapping of the am (Gdh) Locus of Neurospora

Utilizing a combination of flanking marker analysis and deletion mapping we have constructed a fine structure map of the am locus which includes 63 point mutants and ten unique deletions. Positions of point mutants can be rapidly assigned to one of 13 segments within the gene on the basis of crosses to nine deletion strains.     

Studies on the specificity of sperm binding in echinoderm fertilization

Using gametes from the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus, we have evaluated the role of the acrosome reaction and the sperm-egg binding process in the block to interspecific fertilization among echinoids. The results indicate that sperm preinduced to undergo the acrosomal reaction by two different methods still bind to homologous eggs in a species specific manner. These results, taken in conjunction with an earlier study on species specificity of jelly coat induction of the acrosomal reaction (SeGall and Lennarz 1978), indicate that both the acrosome reaction and the sperm binding process contribute to the species specificity of fertilization in S. purpuratus and A. punctulata.     

The Neurospora aab-1 gene encodes a CCAAT binding protein homologous to yeast HAP5.

The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta). A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.     

Epigenetic landscape correlates with genetic subtype but does not predict outcome in childhood acute lymphoblastic leukemia

Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20)(p11–13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving long-term remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes.     

An N-terminal sequence targets and tethers Na+ pump alpha2 subunits to specialized plasma membrane microdomains

Sodium pumps (alphabeta dimers) with the alpha1 isoform of the catalytic (alpha) subunit are expressed in all cells. Additionally, most cells express Na+ pumps with a second alpha isoform. For example, astrocytes and arterial myocytes also express Na+ pumps with the alpha2 isoform. The alpha2 pumps localize to plasma membrane (PM) microdomains overlying "junctional" sarco-/endoplasmic reticulum (S/ER), but the alpha1 pumps are more uniformly distributed. To study alpha2 targeting, we expressed alpha1/alpha2 and alpha2/alpha1 chimeras and 1-90 and 1-120 amino acid N-terminal peptides in primary cultured mouse astrocytes. Immunocytochemistry revealed that alpha2/alpha1 (but not alpha1/alpha2) chimeras markedly reduced native alpha2 (i.e. were "dominant negatives"). N-terminal (1-120 and 1-90 amino acids) alpha2 (and alpha3), but not alpha1 peptides also targeted to the PM-S/ER junctions and were dominant negative for native alpha2 in astrocytes and arterial myocytes. Thus alpha2 and alpha3 have the same targeting sequence. Ca2+ (fura-2) signals in astrocytes expressing the 1-90 alpha2 peptide were comparable to signals in cells from alpha2 null mutants (i.e. functionally dominant negative): 1 microM ATP-evoked Ca2+ transients were augmented, and 100 nM ouabain-induced amplification was abolished. Amino acid substitutions in the 1-120 alpha1 and alpha2 constructs, and in full-length alpha1, revealed that Leu-27 and Ala-35 are essential for targeting/tethering the constructs to PM-S/ER junctions.     

The origin and evolution of <Emphasis Type="Italic">ARGFX</Emphasis>homeobox loci in mammalian radiation

Background  

Many homeobox genes show remarkable conservation between divergent animal phyla. In contrast, the ARGFX (Arginine-fifty homeobox) homeobox locus was identified in the human genome but is not present in mouse or invertebrates. Here we ask when and how this locus originated and examine its pattern of molecular evolution.