Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.     

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.     

Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells

A cDNA clone, pHGR21 encoding poly-ubiquitin, was isolated from a human ovarian granulosa cDNA library. This clone contained three complete, and part of a fourth, ubiquitin coding sequence joined head to tail with no spacer sequences. Northern analysis employing a restriction fragment comprising a complete ubiquitin coding unit indicated the existence of two mRNA species of 1.1kb and 2.8kb. Sequence comparison of pHGR21 with the known two human ubiquitin genes revealed differences to the human ubiquitin-3 repeat gene but significant homology to the human ubiquitin-9 repeat gene. The untranslated 3'-region and the adjacent ubiquitin coding repeat were found to be identical to that of the human ubiquitin-9 repeat gene. The other 3 ubiquitin coding repeats were of close homology to the fourth ubiquitin coding repeat of the human ubiquitin-9 repeat gene. These findings suggest the existence of yet another human poly-ubiquitin gene.     

Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse

Laminin, a substrate adhesion molecule in vertebrates, is a large glycoprotein complex in basement membranes that promotes cell adhesion, cell migration, and neurite outgrowth. Here we report on the cloning of the genes encoding the three subunits of Drosophila laminin. Sequence analysis of cDNA clones encoding the Drosophila B1 chain reveals a multidomain structure similar to that of its mouse homolog. The Drosophila sequence has only 25% amino acid identity with the mouse sequence in domains I, II, and IV. However, in one of the putative collagen-binding regions (domain VI) and the two cysteine-rich domains of EGF-like repeats (domains III and V), the amino acid identity between these two evolutionarily distant species jumps to 55%. Moreover, the number, length, and unique amino acid sequences of each of the 13 EGF-like repeats are highly conserved between Drosophila and mouse, suggesting that each may serve a unique function in protein-protein interactions.     

Cloning of differentially expressed genes in highly and low metastatic rat osteosarcomas by a modified cDNA-AFLP method.

To identify differentially expressed genes between highly and low metastatic rat transplantable osteosarcomas, we applied a modified AFLP (amplified fragment length polymorphisms) method for cDNA subtraction. The specific point of our modification is selective amplification using suppression PCR technique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducibility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology with known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement membrane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologue of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effectors of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and proliferative markers may be important for metastasis of osteosarcomas.     

Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila

Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

The mammalian Odz gene family: homologs of a Drosophila pair-rule gene with expression implying distinct yet overlapping developmental roles

The Drosophila pair-rule gene odz (Tenm) has many patterning roles throughout development. We have identified four mammalian homologs of this gene, including one previously described as a mouse ER stress response gene, Doc4 (Wang et al., 1998). The Odz genes encode large polypeptides displaying the hallmarks of Drosophila Odz: a putative signal peptide; eight EGF-like repeats; and a putative transmembrane domain followed by a 1800-amino-acid stretch without homology to any proteins outside of this family. The mouse genes Odz3 and Doc4/Odz4 exhibit partially overlapping, but clearly distinct, embryonic expression patterns. The major embryonic sites of expression are in the nervous system, including the tectum, optic recess, optic stalk, and developing eye. Additional sites of expression include trachea and mesodermally derived tissues, such as mesentery, and forming limb and bone. Expression of the Odz2 gene is restricted to the nervous system. The expression patterns suggest that each of the genes has its own distinct developmental role. Comparisons of Drosophila and vertebrate Odz expression patterns suggest evolutionarily conserved functions.     

Isolation and characterization of a rice cDNA which encodes a ubiquitin protein and a 52 amino acid extension protein

We isolated a rice cDNA clone encoding the ubiquitin protein fused to a ribosomal protein. This clone encodes a single ubiquitin polypeptide and extension protein of 53 amino acids. This extension protein shows a high degree of homology with those of the yeast ubil or ubi2 gene, both of which encode the same protein. Northern blot analysis suggested that the expression pattern of this gene is more similar to other ribosomal protein genes not linked to ubiquitin protein than to the polyubiquitin gene.     

Isolation and characterization of the gene for myosin light chain two of Drosophila melanogaster

A recombinant lambda-phage DNA clone containing Drosophila melanogaster sequences encoding the gene for myosin light chain (MLC) two has been isolated from a library of randomly sheared DNA. The Drosophila MLC2 gene is located in region 99E1-3 on the right arm of chromosome 3, several bands removed from the site reported for the other myosin light chain gene at 98B. The MLC2 sequence at 99E1-3 appears to encode all of the isoforms of Drosophila MLC2. The polypeptide encoded at 99E was identified as MLC2 by the following criteria: the in vitro translation product is identical in size to MLC2 isolated from Drosophila muscle, and on two-dimensional gels the in vitro translation product can be separated into two or more peptides that co-migrate with isoforms of larval and thoracic MLC2. RNA encoding the polypeptide was detected in embryos only after the onset of muscle differentiation and was also abundant in adult thoracic muscle. The nucleotide sequence of cDNA generated from late embryonic RNA would be translated to yield a protein sequence with multiple regions of homology to vertebrate MLC2. (There are shorter regions of homology to vertebrate MLC1). Like a number of vertebrate muscle proteins, Drosophila MLC2 has an acetylated amino-terminus.     

Search for Drosophila genes encoding a conserved domain present in the achaete-scute complex and myc proteins

Summary Several genes of the achaete-scute complex (ASC) of Drosophila melanogaster encode a 60 amino acids long conserved domain which shares a significant homology with a region of the vertebrate myc proteins. Based on these results, the existence of a family of Drosophila genes that would share both this conserved domain and the neurogenic function of the AS-C has been postulated. To test this proposal, we have searched a D. melanogaster genomic library with a probe that encodes the conserved domain. Only under very low stringency hybridization conditions, clones not belonging to the AS-C cross-hybridized with the probe. Those that gave the strongest signals were characterized. Sequencing of the cross-hybridizing regions showed that they had no significant homology with the conserved domain, the sequence similarity extending at the most for 37 nucleotides. Although our results do not conclusively disprove the existence of a family of AS-C-like genes, they indicate that the conservation of the domain would be lower than that found for shared motifs in other families of Drosophila developmental genes.