Human L1 retrotransposition: insights and peculiarities learned from a cultured cell retrotransposition assay

Long Interspersed Nuclear Elements (L1s or LINEs) are the most abundant retrotransposons in the human genome, and they comprise approximately 17% of DNA. L1 retrotransposition can be mutagenic, and deleterious insertions both in the germ-line and in somatic cells have resulted in disease. Recently, an assay was developed to monitor L1 retrotransposition in cultured human cells. This assay, for the first time, now allows for a systematic study of L1 retrotransposition at the molecular level. Here, I will review progress made in L1 biology during the past three years. In general, I will limit the discussion to studies conducted on human L1s. However, interesting parallels to rodent L1s and other non-LTR retrotransposons also will be discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Ultrastructure of the epididymis of the tammar,Macropus eugenii,and its relationship to sperm maturation

Summary The ductus epididymidis of the tammar is lined by an epithelium composed of principal, mitochondria-rich, apical and basal cells, and intraepithelial leucocytes. The epithelium is structurally differentiated into 6 zones referred to as the initial segment, middle segment (3 subdivisions) and terminal segment (2 subdivisions). The occurrence of the initial, middle and terminal segments corresponds quite closely to the anatomical differentiation of the epididymis into a head, body and tail.The initial segment epithelium in the tammar is lower and has shorter and more slender stereocilia than in other mammals which have been described. Otherwise, the structure of the epithelium has similar characteristics in the tammar to that described in other mammals.Spermatozoa begin to develop the capacity for motility within the initial segment, but only show structural signs of maturation in the middle segment. The sperm head rotates through 90 degrees in the proximal subdivision of the middle segment. The cytoplasmic droplet is detached and spermatozoa develop the capacity for motility in the middle subdivision of the middle segment. The cytoplasmic droplets are phagocytosed by the epididymal epithelium of the middle segment. Sperm storage appears to be the main function of the terminal segment.     

PCR derived cDNA clones for X-linked phosphoglycerate kinase-1 in a marsupial, the tammar wallaby (Macropus eugenii).

cDNA clones for the X-linked PGK-1 were obtained from a tammar wallaby liver by PCR and sequenced. The PGK-1 gene published here is the consensus sequence of those clones. The sequence represents an open reading frame of 1251 bp. Sequence comparisons to X-linked and autosomal sequences showed the greatest homology with the X-linked PGK-1 genes in eutherian species. This sequence opens the way for studying the paternal X inactivation phenomenon in marsupials and will assist in defining the time course of mammalian evolution.     

The metabolic properties of spermatozoa from the epididymis of the tammar wallaby,Macropus eugenii

The utilization of various substrates by sperm from the cauda epididymidis of the tammar was examined because the major naturally occurring sugar in the semen of this species is N-acetyl-D-glucosamine (NAG) and not fructose, as in eutherian mammals. The sperm displayed a high level of endogenous respiration that supported motility for relatively prolonged periods of time in vitro. They also metabolised exogenous ¹⁴C-labelled glucose, NAG, sucrose, and acetate through glycolytic and/or oxidative processes to produce lactate and ¹⁴CO₂ at varying rates. The rate of uptake of NAG by tammar sperm was about four times greater than that of other substrates. Glucose and/or NAG stimulated the rate of oxygen consumption by about 20%, but acetate stimulated oxygen consumption by more than 40%. The most striking findings were that NAG almost completely inhibited the oxidation of glucose and sucrose by the sperm and depressed the uptake of glucose, 3-O-methylglucose, and sucrose. Acetate oxidation also was inhibited by NAG, but only by about 50%. Tammar sperm generated substantial amounts of free glucose during incubation with NAG, but this and the inhibitory effects of NAG on glucose oxidation were not mimicked by rat sperm. It is proposed that tammar sperm fail to oxidise glucose in the presence of NAG because of the rapid cellular uptake of NAG relative to glucose. Also, the intracellular glucose and acetate liberated from NAG would compete with exogenous glucose for processing in the Embden-Meyerhof and tricarboxylic acid (TCA) cycle pathways. It is also suggested that tammar sperm oxidise sucrose after extracellular hydrolysis into its glucose and fructose components. The biological implications of these metabolic and transport properties of tammar sperm have as yet to be determined. Mol. Reprod. Dev. 49:92–99, 1998. © 1998 Wiley-Liss, Inc.     

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii)

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.     

Exsheathment of the infective larva of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby Macropus eugenii.

The infective larva of L. eugenii is enveloped in two cuticles which are discarded when the larva exsheaths in the sacculated portion of the wallaby's stomach. In vitro larvae exsheathed in a 0·85% solution of sodium chloride at 37°C, buffered to pH 7 with bicarbonate ion and 40% carbon dioxide. Survival was enhanced if the liquid phase contained medium 199 and serum, and exsheathment was quicker if exposure to carbon dioxide was 1 h rather than 1 day or 7 days. As larvae exsheathed, contractions of the pharynx commenced, and medium was ingested, even when larvae were enveloped in both cuticles. The stimuli for exsheathment and the subsequent pattern of events are like those already recognised in some trichostrongyles.     

Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition

LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.     

Identification and Characterization of Rhizot,a Novel LTR Retrotransposon of Rhizopus oryzae and R. delemar

A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.     

X-linked gene expression in metatherian fibroblasts: Evidence from theGpd andPgk-A loci of the Virginia opossum and the red-necked wallaby

Fibroblasts cultured from ear pinna biopsies of Virginia opossums (Didelphis virginiana) and red-necked wallabies (Macropus rufogriseus) were examined electrophoretically to determine the relative expression levels of the maternally and paternally derived alleles at X-linked, enzyme-coding loci. Only the maternally derived allele was expressed at thePgk-A locus in fibroblasts of heterozygousD. virginiana (M. rufogriseus not examined), but fibroblasts of both species exhibited evidence of paternal allele expression a t theGpd locus. Furthermore, the heterozygous G6PD phenotypes in both species were skewed in favor of the maternal gene product, as expected if the paternal allele is only partially (incompletely) expressed. ForM. rufogriseus this result is contrary to a previous finding which suggested equal expression of bothGpd alleles in cultured fibroblasts of this species. The present results suggest that X-linked genes in metatherian fibroblasts are subject to the same kind of determinate, paternal allele inactivation, incomplete at some loci, described previously for X-linked genes in adult tissues and that the pattern of paternal X-linked gene expression in these cells is independent of the patterns in the tissues from which the fibroblasts are derived.The work was supported in part by grants from the National Institutes of Health (Biomedical Research Support Grant RR-05519) and the National Science Foundation (DCB 8516949).     

Development of urine concentrating ability in pouch young of a marsupial,the tammar wallaby (Macropus eugenii)

Summary During the first 28–30 weeks after birth, pouch young of the tammar wallaby (Macropus eugenii) normally produce urine less than 500 mOsm/kg and elevate their urine concentration by less than 20% when dehydrated by about 10% of body weight. The adult tammar, in contrast, can produce urine in excess of 3,000 mOsm/kg. The aim of this study was to determine when the various processes involved in urine concentration become mature in the tammar.Vasopressin was detectable in the pituitary of week-old tammars and pituitary vasopressin content decreased significantly after dehydration. Plasma vasopressin did not vary with age and dehydration was associated with an increase in plasma vasopressin levels. By 15 weeks of age at least, tammar kidney slices were able to bind vasopressin as indicated by a rise in tissue cAMP level following hormone treatment.The sodium and urea content of the renal medulla increased with age and significant gradients of these solutes were established by 25 weeks of age. Pouch young older than 25 weeks showed increased medullary sodium and urea levels following dehydration.The inability of pouch young less than 20 weeks of age to produce a highly concentrated urine does not result from any inadequacy in perception of osmotic stimuli or release of vasopressin by the pituitary or of binding of hormone by the kidney. Rather, it appears to be largely attributable to an insufficient medullary hypertonicity, particularly with respect to urea, which is consequent upon structural immaturity of the loop of Henle.Abbreviations cAMP cyclic AMP adenosine 3,5-monophosphate - AVP arginine vasopressin - LVP lysine vasopressin     

Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70–80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.     

Pulmonary cytochrome P450 enzymes belonging to the CYP4B subfamily from an Australian marsupial,the tammar wallaby (Macropus eugenii)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported the cloning and characterisation of the koala CYP4A15, the first reported member of the CYP4 family from marsupials, and have demonstrated important species differences in CYP4A activity and tissue expression. In the present study, the cloning of CYP4B1 in the wallaby (Macropus eugenii) and their expression across marsupials is described. Rabbit anti-mouse CYP4B1 antibody detected immunoreactive proteins in lung and liver microsomes from all test marsupials, with relative weak signal detected from the koala, suggesting a species-specific expression. Microsomal 2-aminofluorene bio-activation (a CYP4B1 marker) in wallaby lung was comparable to that of rabbit, with significant higher activities detected in wallaby liver and kidneys compared to rabbit. A 1548 bp wallaby lung CYP4B complete cDNA, designated CYP4B1, which encodes a protein of 510 amino acids and shares 72% nucleotide and 69% amino acid sequence identity to human CYP4B1, was cloned by polymerase chain reaction approaches. The results demonstrate the presence of wallaby CYP4B1 that shares several common features with other published CYP4Bs; however the wallaby CYP4B1 cDNA contains four extra amino acid residues at the NH₂-terminal, a fundamentally conserved transmembrane anchor of all eukaryote CYPs.     

Identification and characterization of a cDNA encoding ribosomal protein S12 from Xenopus laevis

The complete nucleotide (nt) sequence of the cDNA clone XL-S12, encoding a Xenopus laevis (XI) homologue of the mammalian ribosomal protein S12, has been determined. The sequence predicts a XI S12 protein of 132 amino acids (aa) with a molecular mass of 14.7 kDa. XI S12 shares 95 and 97% aa sequence identity with the human and murine S12 proteins, respectively. Analysis of nt substitution patterns and rates indicates that S12 is a very highly constrained protein, evolving at an estimated rate of only 0.03 x 10˜9 non-synonymous (protein-altering) substitutions per site per year.     

水稻基因组中一类具有潜在转座活性的Solo—LTR的结构分析和鉴定

在水稻4号染色体两个BAC克隆序列分析中,发现了两个solo-LTR,分别命名为SLTR1和SLTR2。它们分别位于水稻18S rRNA基因和一逆转座子内部。序列比较发现,SLTR1和SLTR2存在着较高的同源性,并与水稻逆转座子RIRE8的LTR序列高度同源,分别为89.1%和70.1%。它们属于一类水稻gypsy类型逆转座子。利用SLTR1和SLTR2与水稻DNA杂交,结果显示两者广泛分布于水稻基因组中,是一类高拷贝重复序列。分别利用SLTR1和SLTR2的两侧特异性序列设计引物进行PCR扩增,结果发现在基因组的相应位置并不存在SLTR1或SLTR2;利用它们两侧被打断基因的特异性片段杂交基因组DNA,得到了同样的结果。这意味着SLTR1和SLTR2来源于基因组的其它位置,并通过某种转座的过程进入18S rRNA基因和另一逆转座子内部。Solo-LTR存在着这种潜在的转座活性,对于进一步研究solo-LTR的来源以及其在基因组进货和基因的表达调控中具有一定的意义。