Characterization of a cDNA clone encoding molluscan insulin-related peptide II of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, C alpha and C beta, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the C alpha peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS.     

Molecular characterization of His-Asp phosphorelay signaling factors in maize leaves: Implications of the signal divergence by cytokinin-inducible response regulators in the cytosol and the nuclei

Genes for histidyl-aspartyl (His-Asp) phosphorelay components (His-containing phosphotransfer proteins, HP, and response regulators, RR) were isolated from Zea mays L. to characterize their function in cytokinin signaling. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4–ZmRR7), 3 type-B RRs (ZmRR8–ZmRR10), and 3 HPs (ZmHP1–ZmHP3) were found in leaves. All type-A RR genes expressed in leaves were up-regulated by exogenous cytokinin. Transient expression of fusion products of the signaling modules with green fluorescent protein in epidermal leaf cells suggested cytosolic and nuclear localizations of ZmHPs, whereas type-B ZmRR8 was restricted to the nucleus. Type-A RRs were localized partly to the cytosol (ZmRR1, ZmRR2, and ZmRR3) and partly to the nucleus (ZmRR4, ZmRR5, and ZmRR6). In the yeast two-hybrid assay, ZmHP1 and ZmHP3 interacted with both cytosolic ZmRR1 and nuclear type-B ZmRRs. In vitro experiments demonstrated that ZmHPs function as a phospho-donor for ZmRRs; turnover rates of the phosphorylated state were tenfold lower in ZmRR8 and ZmRR9 than in ZmRR1 and ZmRR4. These results suggest that the His-Asp phosphorelay signaling pathway might diverge into a cytosolic and a nuclear branch in leaves of maize, and that the biochemical nature of ZmRRs is different in terms of stability of the phosphorylated status.     

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.     

Alpha transducin is present in blue-, green-, and red-sensitive cone photoreceptors in the human retina

Phototransduction in vertebrate rod and cone photoreceptor cells involves G protein-mediated light stimulation of cGMP hydrolysis. Enzymes of the cGMP hydrolysis cascades of rods and cones are products of different genes. Three different classes of cones in the human retina are maximally sensitive to either blue, green, or red light. Distinct opsin genes are expressed in each type of cone. The distribution of cone types in human retina was determined using anti-peptide antibodies that recognize specific amino acid sequences in green/red opsin and blue opsin. These antibodies together with an anti-peptide antibody against Tc alpha were used in double labeling experiments to demonstrate the presence of the Tc alpha peptide in all types of cones. cDNA clones corresponding to human rod and cone transducin alpha subunit (Tr alpha and Tc alpha) genes were isolated. Southern blot analyses of human genomic DNA suggest that there is only one rod T alpha gene but more than one cone T alpha gene. The multiple Tc alpha genes could be closely related genes or different Tc alpha alleles, or one could be a pseudogene.     

Expression and cellular localization of the protein encoded by the 1C7 gene: a recently described component of the MHC

The major histocompatibility complex (MHC) is located on human Chromosome 6 and includes clusters of class I, class II, and class III genes. Centromeric to the class I region is a cluster of genes designated as MHC class IV encoding genes involved in immunity and inflammation, including the 1C7 gene. The human 1C7 gene has several alternatively spliced forms and potentially codes for proteins with at least three unique carboxy termini. 1C7 mRNA in human (h1C7) is present in spleen, tonsil, B and NK cell lines, and with a different splicing pattern in liver. The 1C7 RNA and protein are present at highest levels in the germinal center of the lymphoid follicles in tonsil. The protein is expressed in NKL cells, tonsil, and unexpectedly in brain. In contrast, the mouse 1C7 gene is transcribed in liver but is predicted to be a pseudogene. However, the 1C7 homologue expressed in rat is predicted to have long stretches of amino acids essentially identical to the human protein.     

Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

Light-dependent developmental control of rbcS gene expression in epidermal cells of maize leaves

Regulatory elements of the maize rbcS-m3 gene (a member of the family of genes encoding the small subunit of ribulose bisphosphate carboxylase) that are sufficient for expression of the -glucuronidase (gusA) gene in photosynthetic tissue lead to relatively weak expression of the reporter gene in epidermal cells of green maize leaves when delivered by ballistic gene transfer methods. However, epidermal cells of white, immature segments of maize leaf bases express the same reporter gene strongly. Morphologically, these epidermal cells look undifferentiated and are uniform in size and shape. When cultured for seven days on Murashige-Skoog medium [18], exised leaf base segments expand two- to threefold, and epidermal and guard cells differentiate and mature, regardless of whether or not the tissue is illuminated. Epidermal cells that differentiate in darkness continue to have the capacity to express the rbcS-m3:: gusA reporter gene strongly. However, if the leaf base segments are illuminated after four to five days of expansion in darkness, but not before, these more mature epidermal cells are largely unable to express the same gene. That is, they acquire the characteristics of epidermal cells of green maize leaves with regard to expressing the rbcS-m3 reporter gene after undergoing a developmental program (in light or darkness) in vitro and after being exposed to light. White light but not red is effective. Suppression of expression in maize epidermal cells requires different rbcS-m3 sequences than in mesophyll cells [31].     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

A third human CALC (pseudo)gene on chromosome 11

A genomic locus in man (CALC-III) containing nucleotide sequences highly homologous to both exon 2 and exon 3 of the CALC-I and -II genes, is described in this paper. The CALC-I gene produces calcitonin (CT) (encoded by exon 4) or calcitonin gene-related peptide (CGRP) (encoded by exon 5) in a tissue-specific fashion. The CALC-II gene produces a second human CGRP, but probably not a second CT. The CALC-III gene does not seem to encode a CT- or CGRP-related polypeptide hormone and is probably a pseudogene. Like the other two CALC genes, the CALC-III gene is located on human chromosome 11.     

Two gene families clustered in a small region of the Drosophila genome

Three Drosophila genes that are clustered within 8 X 10(3) bases of DNA at the chromosomal region 44D have been identified and mapped, and the gene cluster entirely sequenced. The three genes are 55 to 60% homologous in DNA sequence. One gene contains an intron in its 5'-proximal protein coding sequence while the other two have none at this position; similarly, another gene has an intron in its 3'-proximal protein coding sequence which is not found in the other genes. All three genes are abundantly expressed together in Drosophila first, second, and early third instar larval stages and in adults, but they are not abundantly expressed in either embryonic, late third instar larval, or pupal stages. This gene family lies 11 X 10(3) bases away from another cluster containing four Drosophila larval cuticle protein genes plus a pseudogene. The cuticle genes are all abundantly expressed throughout third instar larval development. Thus, at least seven protein-coding genes and one pseudogene lie within 27 X 10(3) bases of DNA. Moreover, two small gene families can lie adjacent on a chromosome and exhibit different patterns of developmental regulation, even though individual genes within each clustered family are co-ordinately expressed.     

The organization of the keratin I and II gene clusters in placental mammals and marsupials show a striking similarity

The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.     

The intronless mouse gene for the tissue specific splicing protein SmN is a processed pseudogene containing a stop codon after thirty-one amino acids.

The SmN protein is a component of small ribonucleoprotein particles which is closely related to the ubiquitously expressed splicing proteins SmB and B' but is expressed in only a small number of cells and tissues. We have isolated a mouse SmN-related sequence which lacks introns and contains multiple changes from the SmN cDNA sequence including a stop codon after thirty-one amino acids which would prevent it encoding functional SmN protein. This indicates that this intronless gene is a processed pseudogene and that the functional gene has yet to be isolated. In agreement with this southern blotting of mouse DNA with SmN probes reveals bands, additional to those derived from the pseudogene, which are characteristic of an intron-containing SmN gene. The relationship of the pseudogene to the functional SmN gene and to an intronless SmN-related sequence in the rat genome is discussed.     

Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I. Our observations support the hypothesis that light yellow cells are involved in maintaining the shape of the animal via the regulation of ion- and waterbalance processes and blood pressure.