COM,a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster

ZAM and Idefix are two endogenous retroviruses whose expression is tightly controlled in Drosophila melanogaster. However, a line exists in which this control has been perturbed, resulting in a high mobilization rate for both retroviruses. This line is called the U (unstable) line as opposed to the other S (stable) lines. In the process of analyzing this control and tracing the genetic determinant involved, we found that ZAM and Idefix expression responded to two types of controls: one restricting their expression to specific somatic cells in the ovaries and the other silencing their expression in S lines but permitting it in U lines. While studying this second control in the U or S backgrounds, we found that the heterochromatic locus 20A2-3 on the X chromosome, previously implicated in the regulation of a third retroelement, gypsy, also controlled both ZAM and Idefix. We report here that genetic determinants necessary for endogenous retrovirus silencing occur at the 20A2-3 locus, which we call COM, for centre organisateur de mobilisation. We propose that if this point of control becomes mutated during the life of the fly, it may trigger processes reactivating dormant endogenous retroviruses and thus bring about sudden bursts of mobilization.     

Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.     

Simian immunodeficiency virus in which nef and U3 sequences do not overlap replicates efficiently in vitro and in vivo in rhesus macaques

The nef genes of human immunodeficiency virus and simian immunodeficiency virus (SIV) overlap about 80% of the U3 region of the 3' long terminal repeat (LTR) and contain several essential cis-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (att) sequences required for integration. We inactivated the TPI region in the nef reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silent point mutations. To restore viral infectivity, intact cis-regulatory elements were inserted just downstream of the mutated nef gene. The resulting SIV genome contains U3 regions that are 384 bp shorter than the 517-bp 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences truncates the proviral genome by 350 bp. Nonetheless, it contains all known coding sequences and cis-acting elements. The TPI mutant virus expressed functional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restrictions of overlapping LTR sequences and important cis-acting elements. The genomes of all known primate lentiviruses contain a large overlap between nef and the U3 region. We demonstrate that this conserved genomic organization is not obligatory for efficient viral replication and pathogenicity.     

Ultrastructural features of egg development in oviparae of the vetch aphid, Megoura viciae buckton

In the ovaries of the oviparous morph of the aphid, Megoura viciae, resting oocytes are located in the basal region of each germarium. During previtellogenic egg development, electron-dense spheres appear in the ooplasm. During vitellogenesis a brush border develops at the oolemma, and numerous protein and lipid-like spheres accumulate in the egg cytoplasm. Follicle cells are of two morphologically distinct types, termed 'type 1' and 'type 2' follicle cells. Unlike the more numerous 'type 1' cell, 'type 2' cells do not become patent. The acellular tunica propria exterior to follicle cell apices remains intact throughout egg development. During late vitellogenesis symbiont invasion of eggs takes place via 'receptor' cells encircling the pedicel at the posterior egg pole. These cells shrink and/or degenerate to create intercellular spaces that facilitate symbiont transmission. The end of vitellogenesis is marked by vitelline membrane formation and secretion of the chorionic layers, at which time the next egg in the ovariole undergoes final stages of previtellogenic growth and enters vitellogenesis.     

Idefix insulator activity can be modulated by nearby regulatory elements

Insulators play important roles in controlling gene activity and maintaining regulatory independence between neighbouring genes. In this article, we show that the enhancer-blocking activity of the insulator present within the LTR retrotransposon Idefix can be abolished if two copies of the region containing the insulator—specifically, the long terminal repeat (LTR)—are fused to the retrotransposon's 5′ untranslated region (5′ UTR). The presence of this combination of two [LTR-5′ UTR] modules is a prerequisite for the loss of enhancer-blocking activity. We further show that the 5′ UTR causes flanking genomic sequences to be displaced to the nuclear periphery, which is not observed when two insulators are present by themselves. This study thus provides a functional link between insulators and independent genomic modules, which may cooperate to allow the specific regulation of defined genomic loci via nuclear repositioning. It further illustrates the complexity of genomic regulation within a chromatic environment with multiple functional elements.     

Cis-acting sequences which regulate expression of the Sgs-4 glue protein gene of Drosophila.

The Sgs-4 glue protein gene of Drosophila is expressed only in third-instar larval salivary glands. Previous work suggests that a regulatory region lies 5' and remote to the gene, as indicated by a region of tissue-specific DNase I hypersensitivity and by underproducing mutants with DNA lesions in the hypersensitive region. Here we demonstrate by germ line transformation of cloned fragments containing Sgs-4 that the sequences between 840 bp 5' and 130 bp 3' to the gene are sufficient for Sgs-4 activity. When 5' sequence was removed to -392, activity was eliminated, thereby verifying the existence of essential sequences far upstream. Fragments that are active include, in addition to the capacity for normal levels of expression, three other cis-acting regulatory activities: developmental timing, tissue specificity, and dosage compensation. In contrast, the fragments tested did not specify formation of the puff with which Sgs-4 is normally associated. As shown by chromosomal rearrangements, the region required for puffing is limited to 16-19 kb surrounding the gene.     

Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated.

In French bean, the glycine-rich cell wall protein GRP 1.8 is specifically synthesized in the vascular tissue. To identify cis-acting sequences required for cell type-specific synthesis of GRP 1.8, expression patterns of fusion gene constructs were analyzed in transgenic tobacco. In these constructs, the uidA (beta-glucuronidase) gene was placed under control of 5' upstream deletions as well as internal deletions of the GRP 1.8 promoter. Four different cis-acting regulatory regions, SE1 and SE2 (stem elements), a negative regulatory element, and a root-specific element, were found to control the tissue-specific expression. Deletion of the negative regulatory element resulted in expression of the uidA gene in cell types other than vascular cells. The SE1 region was essential for expression in several cell types in the absence of further upstream regulatory sequences. Full-length promoters having insertions between the negative regulatory element and SE1 strongly expressed the gene in nonvascular cell types in stems and leaves. Thus, vascular-specific expression of the GRP 1.8 promoter is controlled by a complex set of positive and negative interactions between cis-acting regulatory regions. The disturbance of these interactions results in expression in additional cell types.     

Enhancer Activity of Solitary Long Terminal Repeat of Human Endogenous Retrovirus K

The transient expression of the luciferase reporter gene was used to detect the tissue-specific enhancer activity of the solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K). The LTR was previously mapped to the 19q13.2 locus. It contains a number of potential regulatory elements including TATA box, binding sites for some nuclear factors, and a polyadenylation signal. However, an analysis of the genomic sequences close to the LTR did not reveal any known genes or the expressed sequences (EST), whose functioning could be regulated by this LTR. The enhancer activity can be preserved in the solitary LTR due to its involvement in the long-range control of genome functioning or by the absence of functional disruptive mutations within the human-specific LTR, because it is of a relatively young evolutionary age.     

Multiple enhancer domains in the 3'' terminus of the Prague strain of Rous sarcoma virus.

The 3' terminal region of the Prague strain of Rous sarcoma virus (PrRSV) contains at least three distinct domains that comprise two functional enhancer elements. Two of these domains (designated B and C) are found in the U3 region of the 3' long terminal repeat (LTR) while the third (designated A) is located in the sequences immediately preceding the LTR termed XSR sequences. Combinations of adjacent domains [e.g., (A + B or B + C)] are capable of activating the expression of the SV40 early promoter (21 bp repeats and TATA box) coupled to coding sequences from the prokaryotic gene chloramphenicol acetyltransferase (CAT) while a single domain is inactive. Furthermore, duplication or triplication of the central domain B restores activity. The related, Schmidt-Ruppin, strain of RSV, contains an almost identical 3' LTR element, but differs in the enhancer sequences immediately preceding the 3' LTR. A model is presented in which the sequence differences may contribute to the difference in disease spectrum of transformation defective (td) variants of these viruses.     

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.     

Retroelement genome painting: cytological visualization of retroelement expansions in the genera Zea and Tripsacum

Divergence of abundant genomic elements among the Zea and Tripsacum genera was examined cytologically and a tool kit established for subsequent studies. The LTR regions from the CRM, Huck, Grande, Prem1, Prem2/Ji, Opie, Cinful-1, and Tekay retroelement families were used as FISH probes on mitotic chromosome spreads from a "trispecies" hybrid containing chromosomes from each of three species: Zea mays (2n = 20), Z. diploperennis (2n = 20), and Tripsacum dactyloides (2n = 36). Except for Tekay, which painted both Zea and Tripsacum chromosomes with nearly equal intensity, the retroelement probes hybridized strongly to the Zea chromosomes, allowing them to be distinguished from those of Tripsacum. Huck and Grande hybridized more intensely to maize than to Z. diploperennis chromosomes. Tripsacum genomic clones containing retroelement sequences were isolated that specifically paint Tripsacum chromosomes. The retroelement paints proved effective for distinguishing different genomes in interspecific hybrids and visualizing alien chromatin from T. dactyloides introgressed into maize lines. Other FISH probes (180-bp knob, TR-1, 5S, NOR, Cent4, CentC, rp1, rp3, and alpha-ZeinA) could be simultaneously visualized with the retroelement probes, emphasizing the value of the retroelement probes for cytogenetic studies of Zea and Tripsacum.