Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum).

One of several functions described for the Fc receptor is regulation of IgG isotype transport into milk. The first marsupial homologues of the Fc receptor heavy and light chains, FcRn and beta-2 microglobulin, from the brushtail possum have been cloned and characterised. The level of FcRn mRNA in the possum mammary gland was highest at the start of lactation, and decreased slowly thereafter. Expression of FcRn mRNA did not increase during the switch phase when the concentration of IgG in milk is highest. In contrast, the level of beta-2 microglobulin mRNA in the mammary gland increased during the switch phase when milk IgG concentration also increases. This correlation between beta-2 microglobulin mRNA expression in the mammary gland with the time of active IgG-transfer into milk was also observed in the bovine and murine mammary gland. This suggests that expression of the Fc receptor in the mammary gland is controlled by the expression of beta-2 microglobulin and that its expression is upregulated during the period of highest IgG-transfer into milk.

Immunological protection of the vulnerable marsupial pouch young: two periods of immune transfer during lactation in Trichosurus vulpecula (brushtail possum).

Marsupial young are born with an underdeveloped immune system and are dependent upon passively acquired immune protection provided by the mother's milk. Colostrum and milk samples were collected from the brushtail possum throughout lactation and the concentration of secretory IgA (sIgA), IgG and transferrin was determined by Western blotting. Two periods of immune transfer were identified. The first, a colostral phase, occurs immediately after birth and involves sIgA, IgG and transferrin. During the early lactation stage, pouch young receive milk of a unique composition as they undergo developmental changes in the pouch that occur in utero for eutherian mammals. At the end of this external gestation, the composition of the milk changes (switch phase) to resemble that of eutherian mammals in the late lactation phase. The second transfer of immunity consists of IgG and transferrin, and occurs during the switch phase prior to maturation of the immune response.

Two stages of increased IgA transfer during lactation in the marsupial, trichosurus vulpecula (Brushtail possum).

The polymeric Ig receptor (pIgR) and J chain molecules are involved in the transfer of IgA across the mammary gland epithelia into milk. The J chain binds two IgA molecules to form dimeric IgA, and the pIgR transports this complex through epithelial cells. We report here the cloning of the first marsupial homologues for the pIgR and J chain from the brushtail possum. Marsupial young are born after a short gestation and are less developed than eutherian newborn. The pouch young is completely dependent on milk as its sole source of nutrition during early lactation and this phase can be considered to be equivalent to an external gestation. Two periods of increased expression of pIgR, J chain, and IgA heavy chain mRNAs were observed in the mammary gland during lactation. The first occurs for a brief period after birth of the pouch young and is likely to reflect IgA transfer via the colostrum. The second period of increased expression, which is unique to marsupials, occurs after the early lactation period and just before young exit the pouch. We propose that this represents a second colostral-like phase at the end of the external gestation.

Cloning and expression of the transferrin and ferritin genes in a marsupial, the brushtail possum (Trichosurus vulpecula).

Transferrin and ferritin cDNAs have been isolated and characterised from the common brushtail possum (Trichosurus vulpecula), the first marsupial examples of these genes. The transferrin cDNA encodes a 711 amino acid pre-protein which shows high levels of amino acid identity with eutherian transferrins (58-60%) and lactoferrins (54-56%). Phylogenetic analysis suggests that the possum transferrin has evolved independently along a pathway distinct from that of the eutherian transferrins and lactoferrins. Possum H-ferritin is a 182 residue protein which shares 86-94% amino acid identity with mammalian, avian and amphibian sequences. Ferritin mRNA was detected in all tissues tested, whereas transferrin was highly expressed in possum liver and mammary gland, and at lower levels in heart, testis and lung. In the possum mammary gland, ferritin mRNA was expressed throughout lactation with higher levels during the first 30 days which coincides with the high iron concentration of milk at this time. The transferrin gene was differentially expressed during lactation with peak mRNA levels detected during the first 6 days of lactation and after day 106 throughout late lactation. The pattern of transferrin mRNA expression in the mammary gland was identical to that of another whey protein, the late lactation protein, suggesting that the transcription of these genes may be regulated by a similar mechanism in this tissue.

A unique isoform of phospholipase Cbeta4 highly expressed in the cerebellum and eye.

We report a unique isoform of PLCbeta4 in rat, PLCbeta4c, that has an additional 37-nucleotide exon inserted between nucleotides 3459-3460 of the previously published PLCbeta4a coding sequence. This insertion results in replacement of 22 amino acid residues at the carboxyl terminal tail of PLCbeta4a with 41 unique residues. A human EST for PLCbeta4 also contains this exon and this exon was mapped to within a 5.5 kb intron of the human PLCbeta4 gene. PLCbeta4c is the third PLCbeta4 isoform to be identified which has a unique carboxyl-terminal tail. PLCbeta4b differs from PLCbeta4a by truncation 162 amino acid residues from the carboxyl terminus which are replaced with 10 distinct amino acid residues. Reverse transcription-polymerase chain reaction experiments show that both PLCbeta4a and PLCbeta4c mRNA are expressed throughout the rat brain and that PLCbeta4c mRNA is highly expressed in the eye and cerebellum. RNase protection assays demonstrate that both PLCbeta4a and PLCbeta4c transcripts are abundant in the cerebellum. The different carboxyl terminal tails of PLCbeta4 isoforms may allow for differential targeting and subcellular localization, contributing to regulation of PLC beta4-mediated signal transduction.

Interaction of eye protein kinase C and INAD in Drosophila. Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies.

Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation. eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains. We previously demonstrated the direct association between the third PDZ domain of INAD with TRP in addition to the carboxyl-terminal half of INAD with the last three residues of NORPA. In this work, the molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays. We show that the second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile. The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC. In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive. Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated. These double mutants display a more severe response inactivation than either of the single mutants. Based on these findings, we conclude that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex.

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions.

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.

Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.

The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli.

The number of molecules of release factor-1 (RF-1) and release factor-2 (RF-2) per Escherichia coli cell grown at various rates was determined using quantitative Western blotting of total solubilized cell protein. The number of RF-1 molecules per cell increased from 1200 to 4900, and of RF-2 from 5900 to 24,900 as growth rates increased from 0.3 to 2.4 doublings per hour. The cellular concentration of the release factors, and therefore efficient termination of protein synthesis is maintained by the increased expression of both RFs as growth rate increases. The expression of both release factors RF-1 and RF-2 is co-ordinated with that of the rest of the translational apparatus, although the increases are less for RF than that for the ribosomes under the same conditions. A significant proportion of the RF pool was found associated with the ribosome fraction. The percentage of ribosomes containing an RF molecule increased from 21 to 33% as the translational rate increased over the growth rate range. Since the cellular concentration of the release factors and their specific activity does not vary significantly with growth rate, this can not provide for an increase in the rate at any of the steps of termination. The postulated strong stop signals, UAAU and UAAG, in genes that are highly expressed at fast growth rates, may result in an increase in the termination rate as a consequence of increased efficiency of decoding by RFs.

Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA.

Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA. The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured. With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition. The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence. The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage. The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state. Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal.

Release factor-dependent false stops are infrequent in Escherichia coli.

We have estimated the frequency of release factor dependent events in which a sense codon is mistakenly translated as a stop codon. We refer to this event as a "false stop". In order to facilitate the measurement of false stop freqeuncies we have used a plasmid expression system to increase individually the cellular levels of release factor (RF) I a and of release factor (RF) 2. We were then able to measure the loss of translational processivity with the aid of a lacZ processivity assay at different concentrations of the release factors. We find that a 30- to 40-fold increase of the RF1 concentration reduces lacZ processivity from 0.6 to 0.3. Assuming that the processivity loss is due only to false stops and that the RF1 overproduction data can be extrapolated back linearly to the normal RF1 concentration in the cell, this corresponds to a false stop frequency close to 10(-5) per codon in the presence of normal amounts of RF1. Furthermore, a threefold increase of the RF2 concentrations had no measurable effect on the processivity of the lacZ gene. Our data suggest that false stops are relatively infrequent compared to the incidence of other translation errors.

Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement.

The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation. We have constructed fusions between the genes for RF-2 and E. coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable. The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site. The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release). These are the first examples of release factor mutants selectively active for only one of these function. In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis. This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.

Frameshifting in the synthesis of Escherichia coli polypeptide chain release factor two on eukaryotic ribosomes.

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. The nucleotide sequence preceding the in-phase stop codon within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that this pairing triggers the frameshift event. In vitro production of RNA coding for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence) region. The prokaryotic RF-2 gene and the gene for the other release factor, RF-1, which does not contain an in-frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA. These RF-1 and RF-2 RNAs both synthesized a major product of Mr approximately 45,000 when translated in vitro within reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules. The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site. The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system. Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of that of RF-1 RNA in the reticulocyte system. This was similar to the relative synthesis of the two proteins in a plasmid-DNA-directed prokaryotic transcription/translation system. These results show that in vitro on eukaryotic ribosomes where the Shine-Dalgarno-type interaction is not possible, high efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can still occur.