The initiator tRNA genes of Drosophila melanogaster: evidence for a tRNA pseudogene

We have isolated four segments of Drosophila melanogaster DNA that hybridize to homologous initiator tRNAMet. Three of the cloned fragments contain initiator tRNA genes, each of which can be transcribed in vitro. The fourth clone, pPW568, contains an initiator tRNA pseudogene which is not transcribed in vitro by RNA polymerase III. The pseudogene is contained in a 1.15 kb DNA fragment. This fragment has the characteristics of dispersed repetitive DNA and hybridizes in situ to at least 30 sites in the Drosophila genome. The arrangement of the initiator tRNA genes we have isolated, is different to that of other Drosophila tRNA gene families. The initiator tRNA genes are not clustered nor intermingled with other tRNA genes. They occur as single copies within an approximately 415-bp repeat segment, which is separated from other initiator tRNA genes by a mean distance of 17 kb. In situ hybridization to polytene chromosomes localizes these genes to the 61D region of the Drosophila genome. Hybridization analysis of genomic DNA indicates the presence of 8-9 non-allelic initiator tRNA genes in Drosophila melanogaster.     

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.     

Clustered Genes Require Extragenic Territorial DNA Sequences

This paper is concerned with the basic question as to whether there exists a complex interaction between DNA sequences which have little specific function and functional genes regarding the spatial arrangement of the gene. Since gene clusters are a characteristic and basic feature of gene structure in higher eukaryotes, the size of extragenic DNA sequences surrounding the individual genes of various clustered gene families were compared. The size of the intergenic region, which is composed of the extragenic DNA sequences flanking the 3'-end of one gene and those flanking the 5'-end of the other gene, of the paired genes increases as the genes becomes larger. However, such a gene size-dependent increase is not seen if the total gene size of the paired genes is less than 0.3 kb or greater than 4 kb. The results suggests that a higher eukaryote gene requires extragenic territorial DNA sequences surrounding it, which presumably are necessary to maintain the gene's active functions.     

Structure, Evolution and Developmental Expression of the Silkmoth Chorion Multigene Families

SYNOPSIS. The silkmoth eggshell (chorion) is a complex extracellularstructure, with important physiological functions concerningsurvival of the developing embryo. The chorion proteins areencoded in several families of genes, generated during evolutionby gene duplication and diversification. The gene families arehighly regulated during development, so that more than 100 distinctchorion proteins are produced during specific stages in choriogenesis.Chorion genes are clustered in a single chromosome, and in theaggregate account for more than 350 kb of DNA. They have beencloned and their structure and organization have been studiedby recombinant DNA techniques. I summarize the information currentlyavailable, and relate it to the processes of gene evolutionand regulation of gene expression during development.     

Small subfamily of olfactory receptor genes: structural features, expression pattern and genomic organization.

Olfactory receptors of the OR37 subfamily are characterized by distinct sequence features and are expressed in neurons segregated in a restricted area of the olfactory epithelium. In the present study, we have characterized the complement of OR37-like genes in the mouse. Five OR37-like genes were identified. They reside within only 60kb of DNA on chromosome 4. About 70kb distant from this cluster, two additional olfactory receptor genes are located, which are members of distinct receptor subfamilies. Phylogenetic analysis demonstrated that the two physically linked receptors are closely related to the OR37 subfamily. Studies of gene expression showed that both genes are also expressed in clustered neuron populations located in the typical OR37 region of the epithelium. These data suggest the involvement of locus-dependent mechanisms for the spatial control of OR gene expression.     

Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37° C

We have analyzed the structure of genes encoding the glyoxylate cycle enzyme isocitrate lyase from Brassica napus L. and their expression during embryogeny and postgermination. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA and genomic clones indicated that there are approximately six isocitrate lyase genes in the B. napus genome that can be divided into at least two subfamilies based upon their divergence in 5′ and 3′ untranslated regions. We showed previously that isocitrate lyase mRNA accumulates during late embryogeny and postgermination. Here, we present results which indicate that several isocitrate lyase genes are expressed at both stages of development. First, gene-specific probes were used to show that mRNAs encoded by representatives of both gene subfamilies accumulated in both late maturation stage embryos and in seedlings of B. napus. Second, a single B. napus isocitrate lyase gene, together with 3.5 kb and 1.4 kb of 5′ and 3′ flanking regions, respectively, was expressed in both embryos and seedlings of transgenic tobacco plants. The results indicated that accumulation of isocitrate lyase in late embryogeny and postgermination does not result from the alternate expression of distinct members of the gene family.     

Structure and flanking regions of soybean seed protein genes

We have characterized the structure and flanking region of genes representing two, coordinately expressed, soybean seed protein gene families. One family directs the synthesis of the major storage protean glycinin; the other encodes a 15.5 kd polypeptide of unknown function. DNA blot hybridization experiments showed approximately three, nonallelic genes in the glycinin family and two in the 15 kd protein family, and showed that these families are not selectively amplified or rearranged during embryogeny. R-loop and S1 nuclease mapping studies demonstrated no detectable introns in the 15 kd protein genes but at least one and possibly two in the glycinin genes. No interfamily clustering of these genes occurs within a 10-15 kb chromosomal domain. Nor are they contiguous to other genes expressed at moderate levels during embryogenesis. Each of them, however, is contiguous to a gene expressed at another developmental period in the leaf. These leaf genes encode rare class messages which constitute only 1 X 10(-5%) of the leaf mRNA, or about one molecule per cell. R-loop analysis of two leaf genes showed that one contains no detectable introns while the other possesses at least three. DNA gel blot studies showed that only one of the seed protein genomic clones contains an interspersed repetitive DNA element. Pairwise cross-hybridization studies did not detect any flanking sequences shared by the 15 kd protein, glycinin and leaf genes.     

Identification and molecular analysis of the cuticle protein genes of Dacus oleae

A 11.9 Kb DNA segment of the Dacus oleae genome that contains three cuticle protein genes has been cloned and characterized. These three genes are clustered within 8.04 Kb of DNA; the restriction sites for eight enzymes and the organization of the cuticle genes in this 11.9 Kb cloned fragment were determined. Using this clone as a radioactive probe, it was shown that the three cuticle genes are expressed as poly(A) RNA in the epidermis of late third instar larvae but are not abundantly expressed in other tissues.     

Sequence Identity in an Early Chorion Multigene Family Is the Result of Localized Gene Conversion

The multigene families that encode the chorion (eggshell) of the silk moth, Bombyx mori, are closely linked on one chromosome. We report here the isolation and characterization of two segments, totaling 102 kb of genomic DNA, containing the genes expressed during the early period of choriogenesis. Most of these early genes can be divided into two multigene families, ErA and ErB, organized into five divergently transcribed ErA/ErB gene pairs. Nucleotide sequence identity in the major coding regions of the ErA genes was 96%, while nucleotide sequence identity for the ErB major coding regions was only 63%. Selection pressure on the encoded proteins cannot explain this difference in the level of sequence conservation between the ErA and ErB gene families, since when only fourfold redundant codon positions are considered, the divergence within the ErA genes is 8%, while the divergence within the ErB genes (corrected for multiple substitutions at the same site) is 110%. The high sequence identity of the ErA major exons can be explained by sequence exchange events similar to gene conversion localized to the major exon of the ErA genes. These gene conversions are correlated with the presence of clustered copies of the nucleotide sequence GGXGGX, encoding paired glycine residues. This sequence has previously been correlated with gradients of gene conversion that extend throughout the coding and noncoding regions of the High-cysteine (Hc) chorion genes of B. mori. We suggest that the difference in the extent of the conversion tracts in these gene families reflects a tendency for these recombination events to become localized over time to the protein encoding regions of the major exons.     

Editor's choice: Large,Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome

To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ∼9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains.     

The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase.

We have determined the exon structure of the mouse tyrosinase-related protein-1 (TRP-1) gene. The gene is only 15kb in length, but contains seven introns, in contrast to the tyrosinase gene which is almost 100kb long with only four introns. Only two introns are located in homologous positions in both genes. Intron I of TRP-1 has three alternative 5' splice sites clustered within 21bp, which all splice to the same 3' site. Intron V has a very unusual 5' splice site, which has the dinucleotide GC rather than the conventional GT. We show that as little as 370bp of 5'-flanking DNA is sufficient to direct cell-specific expression of the chloramphenicol acetyl transferase gene. The flanking DNA of TRP-1, unlike tyrosinase, does not contain a TATA box or a CCAAT box. Both mouse genes, however, share an 11bp sequence, also found in human tyrosinase, which we suggest may be a melanocyte-specific promoter element.     

Interspersion of histone and 5S RNA genes in Artemia

Four recombinant lambda phage containing histone genes were selected from a library of Artemia genomic DNA fragments. The histone gene organization of Artemia resembles that of other invertebrates in that all five genes are clustered and repeated in tandem with approximate repeat lengths of 8.5 kb and 9.3 kb. Each recombinant lambda phage isolate hybridizes with five histone mRNAs and unexpectedly also with 5S ribosomal RNA. Hybridization kinetics have shown the number of histone genes to be about 95-100 copies per haploid genome. An identical number of copies was determined for a hybridization probe containing the 5S gene but no histone genes. We have not found any evidence for a separate set of repeated 5S genes outside this histone + 5S block.     

Genetic and biochemical aspects of keratin synthesis by hair follicles

In this review article the data about synthesis and gene regulation of keratin by hair follicles have been summarized. It has been shown that both differentiation of hair follicle matrix cells and normal growth of hair require the coordinated activities of the genes encoding structural proteins. The keratin genes are clustered in families and are usually 5-10 kb in the genome. The separate clusters of two keratin IF gene families and five KAP gene families have been discovered and some of them have been mapped. The close relation between these clusters suggests that the "global" regulatory domains might govern their expression.     

Structure of the rabbit alphas1- and beta-casein gene cluster, assignment to chromosome 15 and expression of the alphas1-casein gene in HC11 cells

Several casein (CSN) genes (CSN1, 2, 10 and alphas2-CSN) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN/BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN/BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN/BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN/BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.