Interspecies comparison of neuroglobin, cytoglobin and myoglobin: sequence evolution and candidate regulatory elements.

Neuroglobin and cytoglobin are two novel members of the vertebrate globin family. Their physiological role is poorly understood, although both proteins bind oxygen reversibly and may be involved in cellular oxygen homeostasis. Here we investigate the selective constraints on coding and non-coding sequences of the neuroglobin and cytoglobin genes in human, mouse, rat and fish. Neuroglobin and cytoglobin are highly conserved, displaying very low levels of non-synonymous nucleotide substitutions. An oxygen supply function predicts distinct modes of gene regulation, involving hypoxia-responsive transcription factors. To detect conserved candidate regulatory elements, we compared the neuroglobin and cytoglobin genes in mammals and fish. The myoglobin gene was included to test if it also contains hypoxia-responsive regulatory elements. Long conserved non-coding sequences, indicative of gene-regulatory elements, were found in the cytoglobin and myoglobin, but not in the neuroglobin gene. Sequence comparison and experimental data allowed us to delimit upstream regions of the neuroglobin and cytoglobin genes that contain the putative promoters, defining candidate regulatory regions for functional tests. The neuroglobin and the myoglobin genes both lack conserved hypoxia-responsive elements (HREs) for transcriptional activation, but contain conserved hypoxia-inducible mRNA stabilization signals in their 3' untranslated regions. The cytoglobin gene, in contrast, harbors both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation.

Expression analysis of neuroglobin mRNA in rodent tissues.

Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the first detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting differential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is sufficient for its putative primary function as an oxygen-supplying protein.

A vertebrate globin expressed in the brain.

Haemoglobins and myoglobins constitute related protein families that function in oxygen transport and storage in humans and other vertebrates. Here we report the identification of a third globin type in man and mouse. This protein is predominantly expressed in the brain, and therefore we have called it neuroglobin. Mouse neuroglobin is a monomer with a high oxygen affinity (half saturation pressure, P50 approximately 2 torr). Analogous to myoglobin, neuroglobin may increase the availability of oxygen to brain tissue. The human neuroglobin gene (NGB), located on chromosome 14q24, has a unique exon-intron structure. Neuroglobin represents a distinct protein family that diverged early in metazoan evolution, probably before the Protostomia/Deuterostomia split.

Molecular evolution of the globin gene cluster E in two distantly related midges, Chironomus pallidivittatus and C. thummi thummi.

We have studied the evolutionary dynamics of a cluster of insect globin genes by comparing the organization and sequence of the gene group in two distantly related species, Chironomus pallidivittatus and C. t. thummi. Although the general architecture of the globin gene cluster has been conserved, we have found an additional, previously undescribed gene (named Cpa F) in C. pallidivittatus which shows signs of accelerated sequence evolution at nonsynonymous codon positions. This new gene is clearly functional, as demonstrated by Northern analysis. Comparison of paralogous and orthologous genes reveals patterns of intraspecific sequence homogenization. The head-to-head-oriented globin 3 and 4 gene pairs in C. t. thummi and the gb 4 gene pair in C. pallidivittatus have been efficiently homogenized, probably by gene conversion, in their promoter and coding regions. Inverted transcriptional orientation seems to favor efficient conversion. The orthologous genes from C. t. thummi and C. pallidivittatus reveal different levels of sequence conservation, ranging from 85.3 to 94.7% amino acid identity. Surprisingly, globin gene E, for which up to now no corresponding protein has been detected in the larval hemolymph of C. t. thummi, shows the highest degree of interspecies sequence conservation. This points to an essential, as yet unknown function of this globin. The usefulness of globin gene comparisons for dating speciation events in Chironomus is discussed.

Transposition of minisatellite-like DNA in Chironomus midges.

Cla elements are a family of tandem repetitive DNA sequences present in the genome of several Chironomus species. Interspersed clusters of Cla elements are widely distributed all over the chromosomes in C. thummi thummi, while they seem to be limited to the centromeric regions in the closely related subspecies C. t. piger. Here we present molecular evidence that this differential distribution is due to a transposition of Cla elements during evolution of the C. t. thummi genome. We have cloned a "filled" integration site (containing a Cla element cluster) from C. t. thummi and the corresponding "empty"genomic site from C. t. piger and other related species. The comparison shows that tandem repetitive elements may be mobilized together with flanking DNA.